Early endosperm promoter eep1

ABSTRACT

This invention relates generally to the field of plant molecular biology. More specifically, this invention relates to methods and reagents for the temporally- and/or spatially-regulated expression of genes, particularly in plant seeds and related female reproductive tissue. The present invention also provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions comprise novel nucleotide sequences for a seed-preferred promoter known as eep1. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a heterologous nucleotide sequence operably linked to one of the promoters of the present invention and regenerating a stably transformed plant from the transformed plant cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior application Ser. No.10/817,483, filed Apr. 2, 2004 now U.S. Pat. No. 7,531,723, which claimsthe benefit of U.S. Provisional Application No. 60/460,718, filed Apr.4, 2003. All prior applications to which benefit is claimed are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of plant molecularbiology. More specifically, this invention relates to methods andreagents for the temporally- or spatially-regulated expression of genesthat affect metabolically effective levels of cytokinins in plants,including seeds and the maternal tissue from which such seeds arise,including female inflorescences, ovaries, female florets, aleurone,pedicel, and pedicel-forming regions.

BACKGROUND OF THE INVENTION

Cytokinins are phytohormones involved in numerous physiologicalprocesses in plants. Plants respond to environmental stresses in part bymodifying the relative balance of active and inactive cytokinins. Forinstance, during times of abiotic stress (which include, but are notlimited to, conditions of drought, density, cold, salinity, and/or soilcompaction), increased cytokinin oxidase activity shifts the balance infavor of inactive cytokinins, leading to decreased plant productivity.(Jones and Setter, (1999) CSSA Special Publication 29:25-42. AmericanSociety of Agronomy, Madison, Wis.) Conversely, targeted manipulation ofthe cytokinin balance in favor of active cytokinins could result inincreased productivity, even under abiotic stress, through mechanismssuch as increased cell division, induction of stomatal opening,inhibited senescence of organs, and/or suppression of apical dominance.(Morris, R. O., (1997) Cellular and Molecular Biology of Plant SeedDevelopment, pp. 117-148. Kluwer Academic Publishers.) In maize subjectto unfavorable environmental conditions, cytokinins have been shown todecrease resulting in reduced seed size, increased tip kernel abortionand decreased seed set. (Cheikh and Jones, (1994) Plant Physiol.106:45-51; Dietrich, et al., (1995) Plant Physiol Biochem 33:327-336).Therefore, these studies show that under stress conditions one approachto improving seed set and seed size would be to maintain the activecytokinin pool above a critical threshold level.

The first naturally occurring cytokinin was purified in 1963 (Letham, D.S., (1963) Life Sci. 8:569-573) from immature kernels of Zea mays andidentified as 6-(4-hydroxy-3-methylbut-trans-2-enylamino) purine, morecommonly known today as zeatin. In the main all naturally occurringcytokinins appear to be purine derivatives with a branched 5-carbon N⁶substitutent. (See, McGaw, B. A., (1987) Plant Hormones and their Rolein Plant Growth and Development, ed. P. J. Davies, Martinus NijhoffPubl., Boston, Chap B3, pp. 76-93, the contents of which areincorporated by reference for purposes of background.) While some 25different naturally occurring cytokinins have been identified, thoseregarded as particularly active are N⁶ (Δ²-isopentenyl) adenosine (iP),zeatin (Z), diHZ, benzyladenine (BAP) and their 9-ribosyl (and in thecase of Z and diHZ, their O-glucosyl) derivatives. However, suchactivity is markedly reduced in the 7- and 9-glucosyl and 9-alanylconjugates. These latter compounds may be reflective of deactivation orcontrol mechanisms.

The metabolism of cytokinins in plants is complex. Multi-stepbiochemical pathways are known for the biosynthesis and degradation ofcytokinins. At least two major routes of cytokinin biosynthesis arerecognized. The first involves transfer RNA (tRNA) as an intermediate.The second involves de novo (direct) biosynthesis. In the first case,tRNAs are known to contain a variety of hypermodified bases (among themare certain cytokinins). These modifications are known to occur at thetRNA polymer level as a post-transcriptional modification. The branched5-carbon N⁶ substituent is derived from mevalonic acid pyrophosphate,which undergoes decarboxylation, dehydration, and isomerization to yieldΔ²-isopentenyl pyrophosphate (iPP). The latter condenses with therelevant adenosine residue in the tRNA. Further modifications are thenpossible. Ultimately the tRNAs are hydrolyzed to their component bases,thereby forming a pool of available free cytokinins.

Alternately, enzymes have been discovered that catalyze the formation ofcytokinins de novo, i.e., without a tRNA intermediate. The ipt geneutilized in the practice of this invention is one such gene. Theformation of free cytokinins is presumed to begin with [9R5′P] iP. Thiscompound is rapidly and stereospecifically hydroxylated to give thezeatin derivatives from which any number of further metabolic events mayensue. Such events include but are not limited to (1) conjugation,incorporating ribosides, ribotides, glucosides, and amino acids; (2)hydrolysis; (3) reduction; and (4) oxidation. While each enzyme in thesepathways is a candidate as an effector of cytokinin levels, enzymesassociated with rate-limiting steps have particular utility in thepractice of this invention.

One such enzyme is isopentenyl transferase (ipt). An isolated geneencoding ipt was described by van Larebeke. et al., (1974) Nature252:169-170; see also Barry, et al., (1984) Proc. Nat'l. Acad. Sci.(USA) 81:4776-4780 and Strabala, et al., (1989) Mol. Gen. Gen.216(2-3):388-394. Isolation of ipt genes in Arabidopsis has also beenreported. (Takei, et al., (2001) J Biol Chem. 276(28):26405-26410;Kakimoto, et al., (2001) Plant Cell Physiol. 42(7):677-685 and WO2002/072818; Sun, et al., (2003) Plant Physiol 131:167-176) Theinvention comprises appropriately modulated expression of ipt genes fromany source, including other species, such as maize.

Based on the demonstrable effects of cytokinins in hundreds ofexperiments across multiple plant species, a transgenic approach toaugment active cytokinins in maize could improve its productivity undernormal and/or abiotic stress conditions. However, simply increasing thepool of active cytokinins does not automatically lead to enhanced plantgrowth. In fact, elevating cytokinin levels has been shown to generatedetrimental effects on plant phenotype.

For example, Smigocki, et al., (1988) Proc. Nat'l. Acad. Sci. (USA)85:5131-5135, employing the ipt gene from A. tumefaciens operably linkedto either the 35S or NOS promoter, showed a generalized effect on shootorganogenesis and zeatin levels. It was noted that the activity of thepromoter controls the degree of morphogenic response observed, andunregulated production of cytokinins can result in unwanted pleiotropiceffects. With the constructs identified above, undesirable effectsincluded complete inhibition of root formation in tobacco, and stuntedcucumber plantlets that did not survive. Smigocki, et al., supra; Klee,et al., (1987) Annual Rev. Plant Physiol. 38:467-486.

Attempts followed to express the ipt gene in a more controlled fashion.Medford, et al., (1989) The Plant Cell 1:403-413 reported placing theAgrobacterium ipt gene under the control of a heat-inducible promoterand expressing same in transgenic rooted tobacco plants. Levels ofcytokinin rose dramatically following heat treatment, and effectsobserved in transgenics included significant reductions in height, xylemcontent, and leaf size. In both tobacco and Arabidopsis, transgenicsdisplayed slower root growth, disorderly root development, and increasedaxillary bud growth relative to wild-type plants. In addition, theexperimental constructs were not satisfactory because the plantsexhibited phenotypes associated with excess cytokinin levels, includingreduced height, leaf area, and stem width, even in the absence ofthermal induction. Further, certain changes were observed in bothwild-type and transgenic plants and could be attributed to the heatinduction per se.

Schmulling, T., et al., (1989) FEBS Letters 249(2):401-406 transformedtobacco with the Agrobacterium ipt gene under control of the Drosophilahsp70 promoter, which provides a very low level of expression at normaltemperatures and a rapid increase in expression after heat shock. Mostheat-shocked transgenic calli were greener, had higher cytokininconcentrations, and grew at a more rapid rate than control calli. Plantsregenerated from the heat-shocked transgenic calli were described as“fairly normal” and cytokinin levels in these plants did not differ fromthose measured in wild-type plants. Plants regenerated from uninducedtransgenic calli did not differ from controls in either plant phenotypeor cytokinin content. A second experiment created callus tissuetransgenic for the ipt gene driven by its native promoter. In shootsregenerated from these calli, high cytokinin levels inhibited rootformation. These shoots, grafted onto wild-type tobacco stems, displayedtiny leaves and a stunted, highly-branched growth habit. Thus,transformation either resulted in negative phenotypic changes or had noimpact.

In PCT Patent Application Publication No. WO91/01323, 7 Feb. 1991, andU.S. Pat. Nos. 5,177,307, and 4,943,674, tomato plants transformed withthe ipt gene linked to fruit-specific promoters (2AII, Z130 and Z70)exhibited modified ripening characteristics. Fruits were described asroughened at immature stages, and as mottled, blotchy, and patchy duringripening. See also, U.S. Pat. No. 6,329,570, which disclosestransformation of cotton with ipt and a seed-tissue-preferred promoterto modify boll set and fiber quality.

In PCT Patent Application Publication No. WO93/07272, the ipt gene wasfused to the chalcone synthase (chs) promoter from Antirrhinum majus andexpressed in potato. Phenotypic alterations of transformants includedincreased tuber yield, plant height and leaf size, thickened stems anddelayed leaf senescence. Wang, et al., (1997) Australian J of Plant Phys24(5):661-672 and 673-683, reported increased cytokinin levels in leaflaminae and upper stems of tobacco transformed with ipt driven by a chspromoter, as well as release of axillary buds, inhibition of rootdevelopment, retardation of leaf senescence, elevation of chlorophylllevels, delay in onset of flowering, retardation of flower development,growth of leafy shoots from the primary root, change in leaf shape,enlarged leaf midribs, enlarged veins, thicker stems, greater nodenumber, and increased transpiration rates. Expression of chalconesynthase genes is complex and regulated by a variety of factors,including light, fungal elicitors, wounding, and microbial pathogens. Inaddition, chs expression may be tissue-preferred, occurring in pigmentedflowers and roots, and developmentally specific, occurring during earlygermination. Ito, et al., (1997) Mol. Gen. Gen. 255:28-37; Shimizu, etal., (1999) Plant Molecular Biology 39(4):785-95.

Additional ipt gene/promoter constructions have been reported.

Smigocki, et al., in WO 94/24848 and U.S. Pat. Nos. 5,496,732 and5,792,934, disclosed a gene construct capable of conferring enhancedinsect resistance comprising a wound-inducible promoter fused to an iptgene. The study was focused on insect resistance and did not reportchanges in plant morphology.

Houck, et al., in U.S. Pat. Nos. 4,943,674 and 5,177,307, disclosedseveral promoters (2AII, Z130 and Z70) coupled with genes encodingenzymes in the cytokinin metabolic pathway, in particular ipt forexpression of such enzymes in tomato fruit.

Amasino, et al., in PCT Patent Application Publication WO96/29858disclosed two senescence-specific promoters, including SAG12, operablylinked to an ipt gene to inhibit leaf senescence in tobacco.Transformants developed normally, with enhanced biomass and flower andseed production, perhaps owing to the extended developmental periodcreated by the delay in senescence. See also, U.S. Pat. Nos. 5,689,042and 6,359,197; Gan, S., et al., (1995) Science 270:1986-1988. Jordi, etal., (2000) Plant, Cell and Environment 23(3):279-289, studied thephysiological effects of the SAG12:ipt construct in tobacco. While olderleaves benefited by retaining chlorophyll, Rubisco, and protein,remobilization of nutrients from older leaves to younger leaves may havebeen reduced, leading to limited photosynthesis in the upper leaves andrestricting potential increases in biomass of these plants, particularlyunder stress conditions.

Roeckel, P., et al., (1997) Transgenic Res. 6(2):133-141 transformedcanola and tobacco with an ipt gene under the control of thedevelopmentally-regulated, seed-specific 2S albumin promoter fromAgrobacterium. While ipt mRNA was found only in seeds, and cytokininlevels were evaluated only in seeds, effects of the construct were notlimited to seeds: tobacco had reduced roots; canola plants were“surprisingly” (p. 139) taller and had more branches and moreseed-bearing structures. However, yield was not affected, nor was leaftype, leaf number, days to first flower, or days to bolting, in eitherspecies.

Transformation of tobacco with ipt linked to a copper-inducible,root-specific promoter provided, in 28 of 31 cases, a controlled systemfor evaluating effects of increased cytokinin production. Morphologicalchanges upon induction included release of apical dominance, increasesin total plant leaf number, and delay of leaf senescence. (McKenzie, etal., (1998) Plant Physiol. 116:969-977) Several transgenic lines,however, exhibited uncontrolled cytokinin expression and a radicallydifferent, undesirable phenotype, lacking root development andelongation of stems.

Ivic, et al., (2001) Plant Cell Reports 20:770-773 reported thatexpression of ipt in transgenic sugarbeet resulted in severe inhibitionof root development, along with undesirable changes in leaf and shootmorphology. Transformed plantlets formed roots slowly or not at all andhad a very low survival rate when transferred to soil.

Sa, et al., (2002) Transgenic Research 11(3):269-278, reported thattransformation of tobacco with ipt from Agrobacterium under the controlof a TA29 promoter, which specifically expresses in anthers, resulted inperturbation in the development of anthers and pollen. About 80% of theT0 transgenic plants exhibited a significant decrease in the rate ofpollen germination, and up to 20% of the T0 transgenic plants weremale-sterile. In addition, abnormal styles and stamens were found in thetransgenic plants.

Such negative effects resulting from directed expression of transgenicIPT were noted in PCT Publication WO 00/52169: “These approaches alsoproduce undesirable side-effects in the plant and, even in cases whereipt or roIC is expressed under the control of tissue-specific promoters,these side-effects are observed in other tissues, presumably because thecytokinin is transported readily between cells and tissues of theplant.” (emphasis added)

Thus, there still exists a need for nucleic acid constructs and methodsuseful in controlling and directing temporally- and spatially-regulatedexpression of cytokinin metabolic genes in plants, including plant seedand those maternal tissues in which seed development takes place, or inmodulating plant sensing of and/or response to cytokinins, in order toimprove plant vigor and yield without such detrimental effects asreduced root development or aberrant shoot morphology. This inventionprovides several such useful nucleic acid constructs and methods tomodulate cytokinin activity in plants, including effective levels ofcytokinin in plant seeds, developing plant seeds, and related maternalreproductive tissues. Further, the need exists for constructs andmethods which can provide said improvements in plant vigor and yieldunder favorable or unfavorable growing conditions. This inventionprovides tools and reagents that allow the skilled artisan, by theapplication of, inter alia, transgenic methodologies, to so influencethe level of cytokinin activity, including the metabolic flux in respectto the cytokinin metabolic pathway in seed. This influence may be eitheranabolic or catabolic, by which is meant the influence may act toincrease the biosynthesis of cytokinin and/or decrease the degradation.A combination of both approaches is also contemplated by this invention.Further combinations may include targeted modulation of expression ofisolated polynucleotides encoding polypeptides involved in cytokininrecognition and cellular response to provide enhanced cytokinin activityas defined herein.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention provide plants,particularly transgenic maize, which have enhanced cytokinin activity,relative to an otherwise isogenic plant, without correspondingdetrimental effects. Said enhancement relative to an otherwise isogenicplant may occur under favorable environmental conditions, unfavorableenvironmental conditions, or both. Enhanced cytokinin activity mayencompass levels of cytokinins in the seed, the developing seed, and thematernal tissues associated with seed development. Alternatively oradditionally, enhanced cytokinin activity may result from improvedperception of, and response to, cytokinins by said plant. Enhancedcytokinin activity may act as a metabolic buffer to ameliorate theeffects of transient stresses, particularly during the lag phase of seeddevelopment, to thus improve corn stress tolerance and yield stability.Enhanced cytokinin activity may also be manifested in improved plantvigor and/or increased seed yield. Such embodiments comprise a nucleicacid construct stably integrated into the genome thereof, said constructcapable of the temporally- or spatially-regulated modulation ofcytokinin levels.

Certain embodiments of the present invention provide transgenic plantlines with heritable phenotypes which are useful in breeding programsdesigned to produce commercial products with improved performance, whichmay include plant vigor, improved seed size, decreased tip kernelabortion and/or increased seed set during favorable or unfavorableenvironmental conditions. Such commercial products are furtherembodiments of the invention.

Some embodiments of the invention provide a fertile transgenic plantcomprising a nucleic acid construct stably integrated into the genomethereof, said construct capable of effecting modulation of cytokininactivity in said plant.

Certain embodiments of the invention provide an isolated recombinant DNAmolecule comprising a promoter directing temporally- orspatially-regulated expression of an operably-linkedcytokinin-modulating gene and optionally comprising one or more enhancerelements from a highly-expressed gene.

In some embodiments the invention provides a method for improving stresstolerance and yield stability in plants, comprising stably introducinginto plant cells a nucleic acid construct capable of effectingmodulation of cytokinin activity, and from said cells, regenerating saidplants with improved stress tolerance and yield stability. Saidconstruct may result in preferential expression of cytokinin modulatinggenes during the lag phase of plant seed development.

Embodiments also provide a method for producing fertile, transgenicplants capable of the regulated expression of a cytokinin modulatinggene in developing seeds, comprising introducing into plant host cells anucleic acid construct capable of preferential temporal and/or spatialexpression of a cytokinin-modulating gene in developing seed and thematernal tissues associated with seed development, under conditionssufficient for the stable integration of the construct into the genomeof said cells, and regenerating and recovering said fertile transgenicplants.

Further embodiments of the invention provide a method for producingfertile, transgenic plants with enhanced vigor, comprising introducinginto plant host cells a nucleic acid construct capable of effectingmodulation of cytokinin activity, under conditions sufficient for thestable integration of said construct into the genome of said cells, andregenerating and recovering said fertile transgenic plants.

In accordance with these aspects of the invention, there are providedisolated nucleic acid molecules encoding cytokinin metabolic enzymes,including mRNAs, cDNAs, genomic DNAs and biologically useful variants,analogs or derivatives thereof, including fragments of the variants,analogs and derivatives. Other embodiments of the invention arenaturally occurring allelic variants of the nucleic acid molecules inthe sequences provided which encode cytokinin metabolic enzymes. Alsoprovided are polypeptides that comprise cytokinin metabolic enzymes aswell as biologically or diagnostically useful fragments thereof, as wellas variants, derivatives and analogs of the foregoing and fragmentsthereof. For example, specifically provided are cytokinin metabolicpolypeptides, particularly ipt (for example, SEQ ID NOS: 1 and 2) andcytokinin oxidase (for example, SEQ ID NOS: 26-37), that may be employedfor modulation of cytokinin levels in seed and related femalereproductive tissues, particularly meristematic regions of femalereproductive tissues.

Certain embodiments of the invention provide methods for producing thepolypeptides of interest, comprising culturing host cells havingexpressibly incorporated therein a polynucleotide under conditions forthe temporal and/or spatial expression of cytokinin metabolic enzymes inseed and related female reproductive tissues, and then optionallyrecovering the expressed polypeptide.

Also provided in certain embodiments are probes that hybridize tocytokinin metabolic enzyme polynucleotide sequences useful as molecularmarkers in breeding programs.

Other embodiments of the invention provide products, compositions,processes and methods that utilize the aforementioned polypeptides andpolynucleotides for research, biological and agricultural purposes.

Other embodiments of the invention provide inhibitors to suchpolypeptides, useful for modulating the activity and/or expression ofthe polypeptides. In particular, there are provided antibodies againstsuch polypeptides.

In certain embodiments of this aspect of the invention there areprovided antibodies against the cytokinin catabolic enzymes. Theantibodies may be selective for the entire class of the cytokinincatabolic enzymes, irrespective of species of origin, as well asspecies-specific antibodies.

Yet other embodiments provide cytokinin enzyme antagonists and agonists.Among preferred antagonists are those which bind to cytokinin catabolicenzymes (e.g., to cytokinin oxidase) so as to inhibit the binding ofbinding molecules, or to destabilize the complex formed between thecytokinin catabolic enzyme and the binding molecule, to prevent furtherbiological activity arising from the cytokinin catabolic enzyme. Amongpreferred agonists are molecules that bind to or interact with cytokininbiosynthetic enzymes so as to stimulate one or more effects of aparticular cytokinin biosynthetic enzyme or which enhance expression ofthe enzyme and which also preferably result in a modulation of cytokininaccumulation.

Effective constructs result in cytokinin modulation within meristematictissues, particularly those within female reproductive tissues,providing the observed improvement in vigor. The invention encompassesthe particular constructs described herein, and other such constructswhich may provide expression of cytokinin-modulating genes to result inimproved plant vigor without significant detrimental effects. In anycase, and without being limited to any particular theory, the modulationof cytokinin activity in the female reproductive tissues of said plant,to result in enhanced plant vigor without significant detrimentaleffects, is claimed.

Expression of isolated DNA sequences in a plant host is dependent uponthe presence of operably linked regulatory elements that are functionalwithin the plant host. Choice of the regulatory sequences will determinewhen and where within the organism the isolated DNA sequence isexpressed. Where continuous expression is desired in all or nearly allcells of a plant throughout development, constitutive promoters areutilized. In contrast, where gene expression in response to a stimulusis desired, inducible promoters are the regulatory element of choice.Where expression in particular tissues or organs is desired, sometimesat specific stages of development, tissue-preferred promoters and/orterminators are used. That is, these regulatory elements can driveexpression in specific tissues or organs, at specific stages. Additionalregulatory sequences upstream and/or downstream from the core sequencescan be included in expression cassettes of transformation vectors tobring about varying levels of expression of isolated nucleotidesequences in a transgenic plant.

Seed development involves embryogenesis and maturation events as well asphysiological adaptation processes that occur within the seed to insureprogeny survival. Developing plant seeds accumulate and storecarbohydrate, lipid, and protein that are subsequently used duringgermination. Generally, the expression patterns of seed proteins arehighly regulated. This regulation includes spatial and temporalregulation during seed development. A variety of proteins accumulate anddecay during embryogenesis and seed development and provide an excellentsystem for investigating different aspects of gene regulation as well asfor providing regulatory sequences for use in genetic manipulation ofplants.

As the field of plant bioengineering develops, and more genes becomeaccessible, a greater need exists for transforming with multiple genes.These multiple exogenous genes typically need to be controlled byseparate regulatory sequences. Some genes should be regulatedconstitutively, whereas other genes should be expressed at certaindevelopmental stages or locations in the transgenic organism.Accordingly, a variety of regulatory sequences having diverse effectsare needed.

Another reason diverse regulatory sequences are needed is thatundesirable biochemical interactions may result from using the sameregulatory sequence to control more than one gene. For example,transformation with multiple copies of a regulatory element may causehomologous recombination between two or more expression systems,formation of hairpin loops resulting from two copies of the samepromoter or enhancer in opposite orientation in close proximity,competition between identical expression systems for binding to commonpromoter-specific regulatory factors, and inappropriate expressionlevels of an exogenous gene due to trans effects of a second promoter orenhancer.

In view of these considerations, a goal in this field has been thedetection and characterization of new regulatory sequences fortransgenic control of DNA constructs.

Isolation and characterization of seed-preferred promoters andterminators that can serve as regulatory elements for expression ofisolated nucleotide sequences of interest in a seed-preferred manner areneeded for improving seed traits in plants. In particular, early kerneldevelopment is a stage critical in drought-induced ear tip abortion.Maintaining an active pool of plant cytokinins has been proven criticalin sustaining kernel growth and development under transient droughtstress. In addition, genes that contribute to stress responses ingeneral, such as those involved in ABA responses, and also genes thatmaintain cell expansion and division, play essential roles inreproductive development under stress. Early stage endosperm has emergedas an important target tissue for transgene expression as it surroundsand nurtures developing embryos. EEP1 and EEP2 promoters address theneed for directing transgene expression in the early endosperm tissue.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-Embryo: This Figure shows that embryo-preferred overexpressionof ipt increases embryo cytokinin levels, particularly ZR and Z9G (rangeof 2 to 8-fold difference). In contrast, Z levels are unchanged and IPARis not detectable at either developmental stage. Abbreviations:Z=zeatin, ZR (or [9R]Z)=zeatin riboside, Z9G (or[9G]Z)=zeatin-9-glucoside, IPA or [9R]iP=isopentenyladenosine, IPAR (or[9R-5′P]iP)=isopentenyladenosine-5′-monophosphate, and DAP=Days AfterPollination.

FIG. 1B-Endosperm: This Figure shows that embryo-preferred iptoverexpression altered endosperm cytokinin levels but less than those inthe embryo (range of only 10 to 30% difference). Abbreviations used asin FIG. 1A.

FIG. 2 presents ear growth rate data for D2F1 hemizygous plants undernon-stress conditions.

FIG. 3 presents grain yield, kernel number, kernel dry mass, and earlength data for D3F1 hemizygous plants under non-stress conditions.

FIG. 4 presents plant height data for D4F3 homozygous plants undernon-stress conditions.

FIG. 5 presents yield data for D4F3 homozygous plants under non-stressconditions.

FIG. 6 provides yield component data for D4F3 homozygous plants undernon-stress conditions.

FIG. 7 provides plant height data for drought-stressed D4F3 plants.

FIG. 8 provides leaf greenness data for drought-stressed D4F3 plants.

FIG. 9 provides yield data for drought-stressed D4F3 plants.

FIG. 10 shows increased plant biomass for event TC15850.

Sequence Listing Description 1 Agro ipt (pnt) 2 Agro ipt (ppt) 3 zag2.14 CaMV35s enhancer 5 ZmMADS = ZAP 6 promoter ckx1-2 7 eep1 8 end2 9 lec110 F3.7 promoter 11 GSP1 primer for eep1 12 GSP 2 primer for eep1 13Primer for eep1 14 Primer for eep1 15 Clontech AP1 primer 16 ClontechAP2 primer 17 tb1 promoter 18 Eep2 promoter 19 trx1 or thxH promoter(thioredoxin H) 20 Zm40 promoter 21 GSP 1 primer for eep2 22 GSP2 primerfor eep2 23 mLIP15 24 ESR promoter 25 PCNA2 promoter 26 ZmCkx2 pnt 27ZmCkx2 ppt 28 ZmCkx3 pnt 29 ZmCkx3 ppt 30 ZmCkx4 pnt 31 ZmCkx4 ppt 32ZmCkx5 pnt 33 ZmCkx5 ppt 34 ZmCkx2 promoter 35 ZmCkx3 promoter 36 ZmCkx4promoter 37 ZmCkx5 promoter 38 Primer for ipt gene isolation 39 Primerfor ipt gene isolation

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe Examples. The explanations are provided as a convenience and not tolimit the invention.

CYTOKININ ACTIVITY, as used herein, encompasses levels of activecytokinins within a plant, as well as the plant's perception of andresponse to cytokinins. Thus, cytokinin biosynthetic enzymes andcytokinin degrading enzymes are examples of enzymes capable ofmodulating cytokinin activity. “Cytokinin-modulating genes” comprisespolynucleotides encoding such enzymes as well as polynucleotidesencoding proteins involved in cytokinin perception and plant response,including transcription factors associated with the cytokinin response.The “active cytokinin pool” refers to the accumulation of activecytokinins at any one time within a cell or plant part or entire plant,as appropriate. Stabilizing the active cytokinin pool may involvedown-regulation of cytokinin degradation or conjugation, orup-regulation of cytokinin biosynthesis.

CYTOKININ METABOLIC ENZYME-BINDING MOLECULE, as used herein, refers tomolecules or ions which bind or interact specifically with cytokininmetabolic enzyme polypeptides or polynucleotides of the presentinvention, including, for example enzyme substrates, cell membranecomponents and classical receptors. Binding between polypeptides of theinvention and such molecules, including binding or interactionmolecules, may be exclusive to polypeptides of the invention, or it maybe highly specific for polypeptides of the invention, or it may behighly specific to a group of proteins that includes polypeptides of theinvention, or it may be specific to several groups of proteins at leastone of which includes a polypeptide of the invention. Binding moleculesalso include antibodies and antibody-derived reagents that bindspecifically to polypeptides of the invention.

CYTOKININ RESPONSIVE COMPONENT, as used herein, generally means acellular constituent that binds to or otherwise interacts with acytokinin resulting in the transmission of an intra- or inter-cellularsignal and eliciting one or more cellular responses to the presence orabsence or fluctuation in the levels of cytokinins.

DEVELOPING PLANT SEEDS, as used herein, generally means the maternalplant tissues which after pollination are capable of giving rise to aplant seed. This maternal plant tissue includes such tissue as femaleflorets, ovaries, aleurone, pedicel, and pedicel-forming region.

DETRIMENTAL effects, as generally understood and as used herein, arethose which are obviously harmful or damaging. Significant detrimentaleffects, in the context of this application, refer to phenotypic changeswhich would contribute to a net negative effect on plant productivity orvigor.

GENE SILENCING refers to posttranscriptional interference with geneexpression. Techniques such as antisense, co-suppression, and RNAinterference (RNAi), for example, have been shown to be effective ingene silencing. (For reviews, seem Arndt and Rank, (1997) Genome40(6):785-797; Turner and Schuch, (2000) Journal of Chemical Technologyand Biotechnology 75(10):869-882; Klink and Wolniak, (2000) Journal ofPlant Growth Regulation 19(4):371-384)

GENETIC ELEMENT, as used herein, generally means a polynucleotidecomprising a region that encodes a polypeptide, or a polynucleotideregion that regulates replication, transcription or translation or otherprocesses important to expression of the polypeptide in a host cell, ora polynucleotide comprising both a region that encodes a polypeptide anda region operably linked thereto that regulates expression. Geneticelements may be comprised within a vector that replicates as an episomalelement; that is, as a molecule physically independent of the host cellgenome. They may be comprised within plasmids. Genetic elements also maybe comprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

GERMPLASM, as used herein, means a set of genetic entities, which may beused in a breeding program to develop new plant varieties.

HIGH CYTOKININ TRANSGENIC, as used herein, means an entity, which, as aresult of recombinant genetic manipulation, produces seed with aheritable increase in cytokinin and/or decrease in auxin.

HOST CELL, as used herein, is a cell which has been transformed ortransfected, or is capable of transformation or transfection by anexogenous polynucleotide sequence. “Exogenous polynucleotide sequence”is defined to mean a sequence not naturally in the cell, or which isnaturally present in the cell but at a different genetic locus, indifferent copy number, or under direction of a different regulatoryelement.

IDENTITY and SIMILARITY, as used herein, and as known in the art, arerelationships between two polypeptide sequences or two polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness between twopolypeptide or two polynucleotide sequences as determined by the matchbetween two strings of such sequences. Both identity and similarity canbe readily calculated (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to thosedisclosed in Carillo, H., and Lipman, D., SIAM (1988) J. Applied Math.,48:1073. Preferred methods to determine identity are designed to givethe largest match between the two sequences tested. Methods to determineidentity and similarity are codified in computer programs. Typicalcomputer program methods to determine identity and similarity betweentwo sequences include: GCG® program package (Accelrys, Inc., San Diego,Calif.; Devereux, J., et al., (1984) Nucleic Acids Research 12(1):387),BLASTP, BLASTN, FASTA and TFASTA (Atschul, S. F. et al., (1990) J. Mol.Biol. 215:403).

ISOLATED, as used herein, means altered “by the hand of man” from itsnatural state; i.e., that, if it occurs in nature, it has been changedor removed from its original environment, or both. For example, anaturally-occurring polynucleotide or a polypeptide naturally present ina living organism in its natural state is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein. Forexample, with respect to polynucleotides, the term isolated means thatit is separated from the chromosome and cell in which it naturallyoccurs. As part of or following isolation, such polynucleotides can bejoined to other polynucleotides, such as DNAs, for mutagenesis, to formfusion proteins, and for propagation or expression in a host, forinstance. The isolated polynucleotides, alone or joined to otherpolynucleotides such as vectors, can be introduced into host cells, inculture or in whole organisms. Introduced into host cells in culture orin whole organisms, such DNAs still would be isolated, as the term isused herein, because they would not be in their naturally-occurring formor environment. Similarly, the polynucleotides and polypeptides mayoccur in a composition, such as media formulations or solutions forintroduction into cells, or compositions or solutions for chemical orenzymatic reactions, which are not naturally occurring compositions,and, therein such polynucleotides or polypeptides remain isolated withinthe meaning of that term as it is employed herein.

LIGATION, as used herein, refers to the process of formingphosphodiester bonds between two or more polynucleotides, which mostoften are double stranded DNAs. Techniques for ligation are well knownto the art and protocols for ligation are described in standardlaboratory manuals and references, such as, for instance, Sambrook, etal., (1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. and Maniatis, et al.,pg. 146, as cited below.

LOW-LEVEL CONSTITUTIVE EXPRESSION refers to gene expression inessentially all tissues of a plant and at most or all stages ofdevelopment, at a level less than that of a gene driven by the CaMV35Spromoter. Low-level constitutive expression of a polynucleotide mayresult from operable linkage to a promoter that normally drives suchexpression, such as F3.7 (SEQ ID NO: 10) or from a combination of apromoter operably linked to a gene the combination of which is furtherin proximity to an enhancer element, such as the CaMV35s enhancer. (See,for example, Mol. Gen. Gen. 261:635-643 (1999)) Promoters drivingexpression preferentially in meristematic tissues, such as zag2.1 (SEQID NO: 3), may also provide a low level of constitutive expression.

OLIGONUCLEOTIDE(S), as used herein, refers to short polynucleotides.Often the term refers to single-stranded deoxyribonucleotides, but itcan refer as well to single- or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others. Oligonucleotides, suchas single-stranded DNA probe oligonucleotides, often are synthesized bychemical methods, such as those implemented on automated oligonucleotidesynthesizers. However, oligonucleotides can be made by a variety ofother methods, including in vitro recombinant DNA-mediated techniquesand expression of DNAs in cells and organisms. Initially,chemically-synthesized DNAs typically are obtained without a 5′phosphate. The 5′ ends of such oligonucleotides are not substrates forphosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP. The 3′end of a chemically-synthesized oligonucleotide generally has a freehydroxyl group and, in the presence of a ligase, such as T4 DNA ligase,readily will form a phosphodiester bond with a 5′ phosphate of anotherpolynucleotide, such as another oligonucleotide. As is well known, thisreaction can be prevented selectively, where desired, by removing the 5′phosphates of the other polynucleotide(s) prior to ligation.

OPERABLY LINKED, as used herein, includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA correspondingto the second sequence. Generally, operably linked means that thenucleic acid sequences being linked are contiguous and, where necessaryto join two protein coding regions, contiguous and in the same readingframe.

PLANT, as used herein, includes reference to whole plants, plant partsor organs (e.g., leaves, stems, roots, etc.), plant cells, seeds andprogeny of same. Plant cell, as used herein, further includes, withoutlimitation, cells obtained from or found in: seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. Plant cells can alsobe understood to include modified cells, such as protoplasts, obtainedfrom the aforementioned tissues. The class of plants which can be usedin the methods of the invention is generally as broad as the class ofhigher plants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants, including, for example,maize, soybean, and canola.

PLASMIDS, as used herein, generally are designated herein by a lowercase p preceded and/or followed by capital letters and/or numbers, inaccordance with standard naming conventions that are familiar to thoseof skill in the art. Starting plasmids disclosed herein are eithercommercially available, publicly available, or can be constructed fromavailable plasmids by routine application of well-known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art. Moreover, those of skillreadily may construct any number of other plasmids suitable for use inthe invention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentfrom the present disclosure to those of skill.

POLYNUCLEOTIDE(S), as used herein, generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas used herein refers to, among others, single- and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions or single-,double- and triple-stranded regions, single- and double-stranded RNA,and RNA that is a mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded, or triple-stranded, or a mixture of single-and double-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically-,enzymatically- or metabolically-modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including inter alia, simple and complex cells.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally-occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, notonly by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques which are well known to the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Amongthe known modifications which may be present in polypeptides of thepresent invention are, to name an illustrative few, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. Such modifications are well known to those of skill andhave been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as, for instance PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York(1993). Many detailed reviews are available on this subject, such as,for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter, et al., (1990) Meth. Enzymol.182:626-646 and Rattan, et al., (1992) Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.663:48-62. It will be appreciated, as is well known and as noted above,that polypeptides are not always entirely linear. For instance,polypeptides may be branched as a result of ubiquitination, and they maybe circular, with or without branching, generally as a result ofposttranslation events, including natural processing events and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods, aswell. Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications may bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli or othercells, prior to proteolytic processing, almost invariably will beN-formylmethionine. During post-translational modification of thepeptide, a methionine residue at the NH₂-terminus may be deleted.Accordingly, this invention contemplates the use of both themethionine-containing and the methionine-less amino terminal variants ofthe protein of the invention. The modifications that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications in large part will be determined by the hostcell post-translational modification capacity and the modificationsignals present in the polypeptide amino acid sequence. For instance, asis well known, glycosylation often does not occur in bacterial hostssuch as, for example, E. coli. Accordingly, when glycosylation isdesired, a polypeptide should be expressed in a glycosylating host,generally a eukaryotic cell. Similar considerations apply to othermodifications. It will be appreciated that the same type of modificationmay be present in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. In general, as used herein, the term polypeptideencompasses all such modifications, particularly those that are presentin polypeptides synthesized by expressing a polynucleotide in a hostcell.

PROMOTER, as used herein, includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells, such as Agrobacterium orRhizobium. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, or seeds or spatially in regions such asendosperm, embryo, or meristematic regions. Such promoters are referredto as “tissue-preferred”. Promoters that initiate transcription only incertain tissue are referred to as “tissue-specific”. A temporallyregulated promoter drives expression at particular times, such asbetween 0-25 days after pollination. A “cell-type-preferred” promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter that is under environmental control and may be inducibleor de-repressible. Examples of environmental conditions that may effecttranscription by inducible promoters include anaerobic conditions or thepresence of light. Tissue-specific, tissue-preferred,cell-type-specific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterthat is active under most environmental conditions and in all or nearlyall tissues, at all or nearly all stages of development.

RECOMBINANT EXPRESSION CASSETTE, as used herein, refers to a nucleicacid construct, generated recombinantly or synthetically, with a seriesof specified genetic elements that permit transcription of a particularnucleic acid in a host cell. The recombinant expression cassette can beincorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA,virus, or nucleic acid fragment. Typically, the recombinant expressioncassette portion of an expression vector includes, among othersequences, a nucleic acid to be transcribed, and a promoter, and mayoptionally comprise additional elements, such as an enhancer.

RELATED FEMALE REPRODUCTIVE TISSUE, as used herein, includes maternalplant tissues, such as female florets, ovaries, aleurone, pedicel, andpedicel-forming region, either pre-pollination or upon pollination.Pre-pollination seed tissues can also be referred to as “grain initials”or “seed initials”.

TRANSFORMATION, as used herein, is the process by which a cell is“transformed” by exogenous DNA when such exogenous DNA has beenintroduced inside the cell membrane. Exogenous DNA may or may not beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell. In prokaryotes and yeasts, for example, the exogenous DNAmay be maintained on an episomal element, such as a plasmid. Withrespect to higher eukaryotic cells, a stably transformed or transfectedcell is one in which the exogenous DNA has become integrated into thechromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail. With reference to polynucleotides, generally, differences arelimited such that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical. Asnoted below, changes in the nucleotide sequence of the variant may besilent; that is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype, a variant will encode a polypeptide with the same amino acidsequence as the reference. In other cases, as noted below, changes inthe nucleotide sequence of the variant may alter the amino acid sequenceof a polypeptide encoded by the reference polynucleotide. Suchnucleotide changes may result in one or more amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence, as discussed below. With reference to variantpolypeptides generally, differences are limited so that the sequences ofthe reference and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more substitutions, additions, deletions,fusions and truncations, which may be present in any combination.

VIGOR of a plant, as used herein, refers to the relative health,productivity, and rate of growth of the plant and/or of certain plantparts, and may be reflected in various developmental attributes,including, but not limited to, concentration of chlorophyll,photosynthetic rate, total biomass, root biomass, grain quality, and/orgrain yield. In Zea mays in particular, vigor may also be reflected inear growth rate, ear size, and/or expansiveness of silk exsertion. Vigormay be determined with reference to different genotypes under similarenvironmental conditions, or with reference to the same or differentgenotypes under different environmental conditions.

YIELD STABILITY, as known in the art and as used herein, refers toconsistent yield performance of a given genotype across environments,including environments of stress.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates, in part, to nucleic acid constructs useful formodulation of cytokinin activity in plants, including the temporaland/or spatial expression of cytokinin genes in seed and related femalereproductive tissue, and to associated polynucleotides and polypeptides;variants and derivatives of these polynucleotides and polypeptides;processes for making these polynucleotides and these polypeptides, andtheir variants and derivatives; agonists and antagonists of thepolypeptides; products comprising these polynucleotides andpolypeptides, and their variants and derivatives; and uses of thesepolynucleotides, polypeptides, variants, derivatives, agonists andantagonists, and uses of the products comprising same. In particular, inthese and in other regards, the invention relates to polynucleotides andpolypeptides of the cytokinin metabolic pathway, including the enzymesipt and cytokinin oxidase and genes encoding same, and their use singlyor in combination with each other and/or in combinations with variousother isolated polynucleotides and polypeptides affecting cytokininactivity. Targeted modulation of expression to improve plant vigor andseed yield is described.

As mentioned above, the invention provides the reagents necessary forthe development of transgenic plants characterized by enhanced cytokininactivity. As used herein, the phrase “cytokinin activity” is a relativeone and refers to the cytokinin activity in a control plant without thecytokinin-affecting transgene as compared to a plant with such afunctioning transgene. The relative levels may also be measuredemploying only the transgenic plant but measured in the presence andabsence of expression of the subject transgene. Accordingly, anystructural gene, the regulated expression of which has the effect ofenhancing cytokinin activity in plants, particularly seeds, is usefulfor the practice of this invention. Genes that direct the expression ofproteins that act to increase the biosynthesis of cytokinin (e.g., iptor tzs) or genes encoding cytokinin degrading enzymes, the expression ofwhich is inhibited, may be used in the practice of this invention.However, the use of other genes is also contemplated by this invention.In addition to genes that affect the absolute levels of cytokinin, genesthat affect the ratio of cytokinin to auxin are also useful.Auxin-lowering genes such as iaa-1 and gene-5 may also be employed inthe practice of this invention. Additionally or alternatively, targetedmodulation of expression of isolated polynucleotides encodingpolypeptides involved in cytokinin recognition and cellular reponse mayprovide enhanced cytokinin activity as defined herein. Combinations ofthese approaches, comprising changes in expression of one or morecytokinin-modulating genes, are also contemplated.

As mentioned above, the present invention relates to novel constructionsof cytokinin metabolic polypeptides and polynucleotides encoding same,among other things, as described in greater detail below. Thepolypeptides particularly useful for the practice of this inventioninclude, but are not limited to, ipt and cytokinin oxidase. The nucleicacids, and fragments thereof, encoding the above-mentioned enzymes areuseful to generate enzyme-producing transgenics. For example, a singlegene or gene fragment (or combinations of several genes) may beincorporated into an appropriate expression cassette (using for examplethe globulin-1 [glb1] promoter for embryo-preferred expression, or the27 kd gamma zein promoter for endosperm-preferred expression in seed)and transformed into corn along with an appropriate selectable marker(such as the BAR and PAT genes). Certain embodiments comprise a promoterdriving expression in female reproductive meristematic tissue operablylinked to a poynucleotide encoding a cytokinin biosynthetic enzyme.Examples of promoters useful in such an embodiment include zag2.1, Zap(also known as ZmMADS), tb1, and PCNA2, as shown in SEQ ID NOS: 3, 5, 17and 25.

In certain situations it may be preferable to silence or down-regulatecertain genes, such as the cytokinin oxidase. Relevant literaturedescribing the application of homology-dependent gene silencing include:Jorgensen, (1990) Trends Biotechnol. 8(12):340-344; Flavell, (1994)Proc. Nat'l. Acad. Sci. (USA) 91:3490-3496; Finnegan, et al., (1994)Bio/Technology 12:883-888; Neuhuber, et al., (1994) Mol. Gen. Genet.244:230-241; Flavell, et al., (1994) Proc. Natl. Acad. Sci. USA91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol. 31:957-973;Johansen and Carrington (2001) Plant Physiol. 126:930-938; Broin, etal., (2002) Plant Cell 14:1417-1432; Stoutjesdijk, et al., (2002) PlantPhysiol. 129:1723-1731; Yu, et al., (2003) Phytochemistry 63:753-763;and U.S. Pat. Nos. 5,034,323, 5,283,184, and 5,942,657. Alternatively,another approach to gene silencing can be with the use of antisensetechnology (Rothstein, et al., (1989) Plant Mol. Cell. Biol. 6:221-246;Liu, et al., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos.5,759,829 and 5,942,657. Methods and constructs for down-regulatingexpression of cytokinin oxidase are described in US provisional patentapplication, Cytokinin Oxidase-Like Sequences and Methods of Use,60/559,252, filed Apr. 2, 2004, and co-pending U.S. utility applicationSer. No. 11/094,917, filed Mar. 31, 2005.

Certain embodiments may comprise both increased cytokinin biosynthesisand reduced cytokinin degradation to result in improved cytokininactivity.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided the isolated polynucleotides of SEQ ID NOS: 26, 28, 30, and 32,which encode the cytokinin metabolic enzyme maize cytokinin oxidase,having the deduced amino acid sequences shown herein as SEQ ID NOS: 27,29, 31, and 33, as disclosed in co-pending provisional application,Cytokinin Oxidase-Like Sequences and Methods of Use, U.S. provisionalpatent application 60/559,252, filed Apr. 2, 2004, and co-pending U.S.utility application Ser. No. 11/094,917, filed Mar. 31, 2005; as well asmaize cytokinin oxidase of SEQ ID NO:38, encoding SEQ ID NO: 39, asdisclosed in U.S. Pat. No. 6,229,066 and WO99/06571. Use of the isolatedpolynucleotide encoding ipt (isopentenyl transferase), as provided atMolecular and General Genetics 216:388-394 (1989) and provided herein asSEQ ID NO: 1, and its deduced amino acid sequence SEQ ID NO: 2, is alsocontemplated by this invention, as is use of other cytokininbiosynthetic genes (e.g., ipt) isolated from other organisms, such asArabidopsis or maize, for example.

In accordance with one aspect of the present invention, there areprovided the isolated Agrobacterium tumefaciens polynucleotide encodingisopentenyl transferase, SEQ ID NO: 1, and its deduced amino acidsequence, SEQ ID NO: 2 (Strabala, et al., (1989) Mol. Gen. Genet. 216,388-394; GenBank Accession X14410); maize Zag2.1 promoter, SEQ ID NO: 3(GenBank X80206); CaMV 35s enhancer, SEQ ID NO: 4; maize Zap promoter,SEQ ID NO: 5 (also known as ZmMADS; U.S. patent application Ser. No.10/387,937; WO 03/078590); maize ckx1-2 promoter, SEQ ID NO: 6 (USpatent publication 2002-0152500 A1; WO 02/0078438); maize eep1 promoter,SEQ ID NO: 7 (U.S. provisional patent application 60/460,718); maizeend2 promoter, SEQ ID NO: 8 (U.S. Pat. No. 6,528,704 and U.S. patentapplication Ser. No. 10/310,191); maize lec1 promoter, SEQ ID NO: 9(U.S. patent application Ser. No. 09/718,754); maize F3.7 promoter, SEQID NO: 10 (Baszczynski, et al., (1997) Maydica 42:189-201; maize tb1promoter, SEQ ID NO: 17 (Hubbarda, et al., (December 2002) Genetics162:1927-1935; maize eep2 promoter, SEQ ID NO: 18; maize thioredoxinHpromoter, SEQ ID NO: 19, U.S. provisional Patent Application60/514,123); maize Zm40 promoter, SEQ ID NO: 20 (U.S. Pat. No. 6,403,862and WO 01/2178); maize mLIP15 promoter, SEQ ID NO: 23 (U.S. Pat. No.6,479,734); maize ESR promoter, SEQ ID NO: 24 (U.S. application Ser. No.10/786,679, filed Feb. 25, 2004); maize PCNA2 promoter, SEQ ID NO: 25(U.S. application Ser. No. 10/388,359 filed Mar. 13, 2003); maizecytokinin oxidases and promoters, SEQ ID NOS: 26-37 (US provisionalpatent application, Cytokinin Oxidase-Like Sequences and Methods of Use,60/559,252, filed Apr. 2, 2004, and co-pending U.S. utility applicationSer. No. 11/094,917, filed Mar. 31, 2005).

The maize gene ZAG2 was isolated based on homology to the ArabidopsisAGAMOUS gene, which directs floral development. (Schmidt, et al., (1993)Plant Cell 5(7):729-737) ZAG2 is normally expressed primarily indeveloping female florets. The ZAG2 coding sequence and approximately2.1 kb of 5′ sequence were deposited in GenBank as accession no. X80206in September 1995. A portion of the ZAG2 5′ region is included herein asSEQ ID NO: 3 and referred to as the ZAG2.1 promoter.

Using the information provided herein, such as the polynucleotidesequences set out below, a polynucleotide of the present inventionencoding cytokinin metabolic enzyme polypeptides may be obtained usingstandard cloning and screening procedures. To obtain the polynucleotideencoding the protein using the DNA sequences given below,oligonucleotide primers can be synthesized that are complementary to theknown polynucleotide sequence. These primers can then be used in PCR toamplify the polynucleotide from template derived from mRNA or genomicDNA isolated from the desired source material. The resulting amplifiedproducts can then be cloned into commercially available cloning vectors,such as the TA series of vectors from InVitrogen. By sequencing theindividual clones thus identified with sequencing primers designed fromthe original sequence, it is then possible to extend the sequence inboth directions to determine the full gene sequence. Such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook, J. in MOLECULAR CLONING, A Laboratory Manual (2nd edition1989 Cold Spring Harbor Laboratory. See, Sequencing DenaturedDouble-Stranded DNA Templates 13.70.

Isolation of ipt Gene:

The isopentenyl transferases (ipts) of the present invention may beobtained from sources including, but not limited to, Zea mays,Agrobacterium, Psuedomonas savastano, Rhodococcus and Erwinia. Thecomplete sequence of an ipt gene is provided in Strabala, T. J., et al.,(1989) Isolation and characterization of an ipt gene from the Ti plasmidBo542, Mol. Gen. Genet. 216:388-94. A copy of such gene can be preparedsynthetically employing DNA synthesis protocols well known to thoseskilled in the art of gene synthesis. Alternatively, a copy of the genemay be isolated directly from an organism harboring an ipt gene, forexample by PCR cloning as described in WO 00/63401, herein incorporatedby reference.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the antisense strand.

The coding sequence that encodes the polypeptide may be identical to thecoding sequence of the polynucleotides shown below. It also may be apolynucleotide with a different sequence, which, as a result of theredundancy (degeneracy) of the genetic code, encodes the polypeptidesshown below. As discussed more fully below, these alternative codingsequences are an important source of sequences for codon optimization.

Polynucleotides of the present invention which encode the polypeptideslisted below may include, but are not limited to, the coding sequencefor the mature polypeptide, by itself; the coding sequence for themature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals, for example), ribosome binding, mRNA stabilityelements, and additional coding sequences which encode additional aminoacids, such as those which provide additional functionalities.

The DNA may also comprise promoter regions that function to direct thetranscription of the DNA encoding heterologous cytokinin-modulatingenzymes of this invention. Heterologous is defined as a sequence that isnot naturally occurring with the promoter sequence. While the nucleotidesequence is heterologous to the promoter sequence, it may be homologous(native) or heterologous (foreign) to the plant host.

Furthermore, the polypeptide may be fused to a marker sequence, such asa peptide, which facilitates purification of the fused polypeptide. Incertain embodiments of this aspect of the invention, the marker sequenceis a hexa-histidine peptide, such as the tag provided in the pQE vector(Qiagen, Inc.) and the pET series of vectors (Novagen), among others,many of which are commercially available. As described in Gentz, et al.,(1989) Proc. Nat'l. Acad. Sci., (USA) 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. The HA tag may also be used to create fusion proteins andcorresponds to an epitope derived of influenza hemagglutinin protein,which has been described by Wilson, et al., (1984) Cell 37:767, forinstance.

In accordance with the foregoing, the term “polynucleotide encoding apolypeptide” as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlycytokinin modulating enzymes having the amino acid sequences set outbelow. The term encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or insertion sequence orediting) together with additional regions that also may contain codingand/or non-coding sequences.

The present invention further relates to variants of the presentpolynucleotides that encode for fragments, analogs and derivatives ofthe polypeptides having the deduced amino acid sequence below. A variantof the polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally-occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.

Among the embodiments of the invention in this regard arepolynucleotides encoding polypeptides having the amino acid sequencesset out below; variants, analogs, derivatives and fragments thereof.

Further in this regard are polynucleotides encoding cytokininbiosynthetic enzyme variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequences below in which several, a few, 1 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Among these are polynucleotides comprising silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the cytokinin biosynthetic enzymes;conservative substitutions; and polynucleotides encoding polypeptideshaving the amino acid sequence below, without substitutions.

Further embodiments of the invention comprise polynucleotides that aregreater than 79%, at least 80%, or at least 85% identical to apolynucleotide encoding a polypeptide having an amino acid sequence setout below, and polynucleotides that are complementary to suchpolynucleotides. Certain embodiments, moreover, are polynucleotideswhich encode polypeptides which retain substantially the same, or evenexhibit a increase in, biological function or activity as compared tothat of the mature polypeptide encoded by the polynucleotides set outbelow.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 80% identity between the sequences.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, often less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. Hybridizationand/or wash conditions can be applied for at least 10, 30, 60, 90, 120or 240 minutes. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding cytokininbiosynthetic enzymes and to isolate cDNA and genomic clones of othergenes that have a high sequence similarity to the genes. Such probesgenerally will comprise between about 15 and 50 bases.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of transgenicplants with modulated cytokinin activity. The polynucleotides of theinvention that are oligonucleotides derived from the sequences below maybe used as PCR primers in the process herein described to determinewhether or not the genes identified herein in whole or in part aretranscribed in cytokinin accumulating tissue.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences, may be an inactive form of the polypeptide.When prosequences are removed, such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Polypeptides

The present invention further relates to polypeptides that have thededuced amino acid sequences below. The polypeptide of the presentinvention may be a recombinant polypeptide, a natural polypeptide or asynthetic polypeptide. In certain embodiments it is a recombinantpolypeptide.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms “fragment,” “derivative” and “analog”,when referring to the polypeptides, mean a polypeptide which retains atleast 90% of, at least 95% of, or essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein that can be activated by cleavage of the proprotein portionto produce an active mature polypeptide. Among the embodiments of theinvention in this regard are polypeptides having the amino acid sequenceof cytokinin modulating enzymes set out below, variants, analogs,derivatives and fragments thereof, and variants, analogs and derivativesof the fragments.

The fragment, derivative or analog of the polypeptides below may be (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be obtained by those of ordinary skill in the art,from the teachings herein.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Arg;and replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequences below, in which several, afew, 1 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, which donot alter the properties and activities of the cytokinin biosyntheticenzymes. Also especially preferred in this regard are conservativesubstitutions. Most highly preferred are polypeptides having the aminoacid sequences below without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and may be purified tohomogeneity.

Vectors, Host Cells, Expression

The present invention also relates to vectors comprising thepolynucleotides of the present invention, host cells that incorporatethe vectors of the invention, and the production of polypeptides of theinvention by recombinant techniques.

Vectors

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors, also may be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host, operably linked to the polynucleotide to beexpressed. Appropriate trans-acting factors are supplied by the host,supplied by a complementing vector or supplied by the vector itself uponintroduction into the host.

In certain preferred embodiments in this regard, the vectors provide forpreferred expression. Such preferred expression may be inducibleexpression or temporally limited or restricted to predominantly certaintypes of cells or any combination of the above. Particularly preferredamong inducible vectors are vectors that can be induced for expressionby environmental factors that are easy to manipulate, such astemperature and nutrient additives. A variety of vectors suitable tothis aspect of the invention, including constitutive and inducibleexpression vectors for use in prokaryotic and eukaryotic hosts, are wellknown and employed routinely by those of skill in the art. Such vectorsinclude, among others, chromosomal, episomal and virus-derived vectors,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids andbinaries used for Agrobacterium-mediated transformations. All may beused for expression in accordance with this aspect of the presentinvention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Useful plant binary vectorsinclude BIN19 and its derivatives available from Clontech. These vectorsare listed solely by way of illustration of the many commerciallyavailable and well-known vectors that are available to those of skill inthe art for use in accordance with this aspect of the present invention.It will be appreciated that any other plasmid or vector suitable for,for example, introduction, maintenance, propagation or expression of apolynucleotide or polypeptide of the invention in a host may be used inthis aspect of the invention, several of which are disclosed in moredetail below.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, aribosome-binding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include atranslation-initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as transcription factors, repressor binding sitesand termination signals, among others. For secretion of the translatedprotein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. Additional enhancers useful in theinvention to increase transcription of the introduced DNA segment,include, inter alia, viral enhancers like those within the 35S promoter,as shown by Odell, et al., (1988) Plant Mol. Biol. 10:263-72, and anenhancer from an opine gene as described by Fromm, et al., (1989) PlantCell 1:977. The enhancer may affect the tissue-specificity and/ortemporal specificity of expression of sequences included in the vector.For example, a construct may comprise the CaMV 35s enhancer (SEQ ID NO:4) in a “head to head” orientation with respect to the zag2.1 promoter(SEQ ID NO: 3) driving ipt (SEQ ID NO: 1).

Termination regions also facilitate effective expression by endingtranscription at appropriate points. Useful terminators for practicingthis invention include, but are not limited to, pinII (See, An, et al.,(1989) Plant Cell 1(1):115-122), glb1 (See, Genbank Accession #L22345),gz (See, gzw64a terminator, Genbank Accession #S78780), and the nosterminator from Agrobacterium.

Among known eukaryotic promoters suitable for generalized expression arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), metallothionein promoters, suchas the mouse metallothionein-I promoter and various plant promoters,such as globulin-1. When available, the native promoters of thecytokinin modulating enzyme genes may be used. Representatives ofprokaryotic promoters include the phage lambda PL promoter, the E. colilac, trp and tac promoters to name just a few of the well-knownpromoters.

With respect to plants, examples of seed-preferred promoters includepromoters of seed storage proteins which express these proteins in seedsin a highly regulated manner (Thompson, et al., (1989) BioEssays,10:108), such as, for dicotyledonous plants, a bean β-phaseolinpromoter, a napin promoter, a β-conglycinin promoter, and a soybeanlectin promoter. For monocotyledonous plants, promoters useful in thepractice of the invention include, but are not limited to, a maize 15 kDzein promoter, a 22 kD zein promoter, a 27 Kd γ-zein promoter (such asgzw64A promoter, see, Genbank Accession #S78780), a waxy promoter, ashrunken-1 promoter, a globulin 1 promoter (see, Genbank Accession#L22344), an Itp2 promoter (Kalla, et al., (1994) Plant Journal6:849-860; U.S. Pat. No. 5,525,716), cim1 promoter (see, U.S. Pat. No.6,225,529) maize end1 and end2 promoters (See, U.S. Pat. No. 6,528,704and application Ser. No. 10/310,191, filed Dec. 4, 2002); nuc1 promoter(U.S. Pat. No. 6,407,315); Zm40 promoter (U.S. Pat. No. 6,403,862); eep1(SEQ ID NO: 7) and eep2 (SEQ ID NO: 18); lec1 (U.S. patent applicationSer. No. 09/718,754); thioredoxinH promoter (U.S. provisional patentapplication 60/514,123); mlip15 promoter (U.S. Pat. No. 6,479,734);PCNA2 promoter, SEQ ID NO: 25; and the shrunken-2 promoter. (Shaw, etal., (1992) Plant Phys 98:1214-1216; Zhong Chen, et al., (2003) PNAS USA100:3525-3530) However, other promoters useful in the practice of theinvention are known to those of skill in the art such as nucellainpromoter (see, C. Linnestad, et al., (1998) Nucellain, A Barley Homologof the Dicot Vacuolar—Processing Proteasem Is Localized in Nucellar CellWalls, Plant Physiol. 118:1169-80, kn1 promoter (see, S. Hake and N.Ori, The Role of knotted1 in Meristem Functions, B8: INTERACTIONS ANDINTERSECTIONS IN PLANT PATHWAYS, COEUR D'ALENE, IDAHO, KEYSTONESYMPOSIA, Feb. 8-14, 1999, at 27.), and F3.7 promoter (Baszczynski, etal., (1997) Maydica 42:189-201; SEQ ID NO: 10). Spatially actingpromoters such as glb1, an embryo-preferred promoter; or gamma zein, anendosperm-preferred promoter; or a promoter active in theembryo-surrounding region (see, U.S. patent application Ser. No.10/786,679, filed Feb. 25, 2004), or BETL1 (see, G. Hueros, et al.,(1999) Plant Physiology 121:1143-1152 and Plant Cell 7:747-57 (June1995)), are particularly useful, including promoters preferentiallyactive in female reproductive tissues, and those active in meristematictissues, particularly in meristematic female reproductive tissues.

The use of temporally-acting promoters is also contemplated by thisinvention. Promoters that act from 0-25 days after pollination (DAP) arepreferred, as are those acting from 4-21, 4-12, or 8-12 DAP. In thisregard, promoters such as cim1 and Itp2 are preferred. Promoters thatact from −14 to 0 days after pollination can also be used, such as SAG12(see, WO 96/29858, Richard M. Amasino, published 3 Oct. 1996) and ZAG1or ZAG2 (see, R. J. Schmidt, et al., Identification and MolecularCharacterization of ZAG1, the Maize Homolog of the Arabidopsis FloralHomeotic Gene AGAMOUS, Plant-Cell 5(7):729-37 (July 1993). See also, SEQID NO: 3).

Useful promoters include maize zag2.1 (SEQ ID NO: 3), Zap (SEQ ID NO: 5,also known as ZmMADS; U.S. patent application Ser. No. 10/387,937; WO03/078590); maize tb1 promoter (SEQ ID NO: 17; see also, Hubbarda, etal., (2002) Genetics 162:1927-1935).

Examples of suitable promoters for generalized expression in plants arethe promoter for the small subunit of ribulose-1,5-bis-phosphatecarboxylase, promoters from tumor-inducing plasmids of Agrobacteriumtumefaciens, such as the nopaline synthase and octopine synthasepromoters, and viral promoters such as the cauliflower mosaic virus(CaMV) 19S and 35S promoters or the figwort mosaic virus 35S promoter.

It will be understood that numerous promoters not mentioned are suitablefor use in this aspect of the invention, are well known and readily maybe employed by those of skill in the manner illustrated by thediscussion and the examples herein. For example, this inventioncontemplates using, when appropriate, the native cytokinin biosyntheticenzyme promoters to drive the expression of the enzyme in a recombinantenvironment.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other prokaryotes. Kanamycinand herbicide resistance genes (PAT and BAR) are generally useful inplant systems.

Selectable marker genes, in physical proximity to the introduced DNAsegment, are used to allow transformed cells to be recovered by eitherpositive genetic selection or screening. The selectable marker genesalso allow for maintaining selection pressure on a transgenic plantpopulation, to ensure that the introduced DNA segment, and itscontrolling promoters and enhancers, are retained by the transgenicplant.

Many of the commonly used positive selectable marker genes for planttransformation have been isolated from bacteria and code for enzymesthat metabolically detoxify a selective chemical agent which may be anantibiotic or a herbicide. Other positive selection marker genes encodean altered target which is insensitive to the inhibitor.

An example of a selection marker gene for plant transformation is theBAR or PAT gene, which is used with the selecting agent bialaphos.Spencer, et al., (1990) J. Theor. Appl'd Genetics 79:625-631. Anotheruseful selection marker gene is the neomycin phosphotransferase II(nptII) gene, isolated from Tn5, which confers resistance to kanamycinwhen placed under the control of plant regulatory signals. Fraley, etal., (1983) Proc. Natl. Acad. Sci. (USA) 80:4803. The hygromycinphosphotransferase gene, which confers resistance to the antibiotichygromycin, is a further example of a useful selectable marker. VandenElzen, et al., (1985) Plant Mol. Biol. 5:299. Additional positiveselectable marker genes of bacterial origin that confer resistance toantibiotics include gentamicin acetyl transferase, streptomycinphosphotransferase, aminoglycoside-3′-adenyl transferase and thebleomycin resistance determinant. Hayford, et al., (1988) Plant Physiol.86:1216; Jones, et al., (1987) Mol. Gen. Genet. 210:86; Svab, et al.,(1990) Plant Mol. Biol. 14:197; Hille, et al., (1986) Plant Mol. Biol.7:171.

Other positive selectable marker genes for plant transformation are notof bacterial origin. These genes include mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz, et al., (1987) Somatic Cell Mol. Genet. 13:67;Shah, et al., (1986) Science 233:478; Charest, et al., (1990) Plant CellRep. 8:643.

Another class of useful marker genes for plant transformation with theDNA sequence requires screening of presumptively transformed plant cellsrather than direct genetic selection of transformed cells for resistanceto a toxic substance such as an antibiotic. These genes are particularlyuseful to quantitate or visualize the spatial pattern of expression ofthe DNA sequence in specific tissues and are frequently referred to asreporter genes because they can be fused to a gene or gene regulatorysequence for the investigation of gene expression. Commonly used genesfor screening presumptively transformed cells include β-glucuronidase(GUS), β-galactosidase, luciferase, and chloramphenicolacetyltransferase. Jefferson, (1987) Plant Mol. Biol. Rep. 5:387; Teeri,et al., (1989) EMBO J. 8:343; Koncz, et al., (1987) Proc. Nat'l Acad.Sci. (USA) 84:131; De Block, et al., (1984) EMBO J. 3:1681. Anotherapproach to the identification of relatively rare transformation eventshas been use of a gene that encodes a dominant constitutive regulator ofthe Zea mays anthocyanin pigmentation pathway (Ludwig, et al., (1990)Science 247:449).

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. The sequence may be inserted in a forward or reverseorientation. Procedures for restriction and ligation that can be used tothis end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseof skill, are set forth in great detail in Sambrook, et al., (1989)MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

A polynucleotide of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention, generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5′ to aribosome binding site. The ribosome-binding site will be 5′ to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiation codon. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal in constructs for use in eukaryotic hosts.Transcription termination signals appropriately disposed at the 3′ endof the transcribed region may also be included in the polynucleotideconstruct.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well-known techniques suitable to expressiontherein of a desired polypeptide. The present invention also relates tohost cells containing the above-described constructs. The host cell canbe a higher eukaryotic cell, such as a plant cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis, et al., (1986) BASIC METHODS IN MOLECULAR BIOLOGY andSambrook, et al., (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS and Bowes melanoma cells; and plantcells. The plant cells may be derived from a broad range of plant types,particularly monocots such as the species of the Family Graminiaeincluding Sorghum bicolor and Zea mays, as well as dicots such assoybean (Glycine max) and canola (Brassica napus, Brassica rapa ssp.).Preferably, plants include maize, soybean, sunflower, safflower, canola,wheat, barley, rye, alfalfa, and sorghum; however, the isolated nucleicacid and proteins of the present invention can be used in species fromthe genera: Ananas, Antirrhinum, Arabidopsis, Arachis, Asparagus,Atropa, Avena, Brassica, Bromus, Browaalia, Camellia, Capsicum,Ciahorium, Citrus, Cocos, Cofea, Cucumis, Cucurbita, Datura, Daucus,Digitalis, Ficus, Fragaria, Geranium, Glycine, Gossypium, Helianthus,Heterocallis, Hordeum, Hyoscyamus, Ipomoea, Juglans, Lactuca, Linum,Lolium, Lotus, Lycopersicon, Majorana, Mangifera, Manihot, Medicago,Musa, Nemesis, Nicotiana, Olea, Onobrychis, Oryza, Panieum, Pelargonium,Pennisetum, Persea, Petunia, Phaseolus, Pisum, Psidium, Ranunculus,Raphanus, Rosa, Salpiglossis, Secale, Senecio, Solanum, Sinapis,Sorghum, Theobroma, Triticum, Trifolium, Trigonella, Vigna, Vitis, andZea.

The promoter regions of the invention may be isolated from any plant,including, but not limited to, maize (corn; Zea mays), canola (Brassicanapus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats, barley, vegetables, ornamentals, and conifers.Preferably, plants include maize, soybean, sunflower, safflower, canola,wheat, barley, rye, alfalfa, and sorghum.

Hosts for a great variety of expression constructs are well known, andthose of skill will be enabled by the present disclosure readily toselect a host for expressing a polypeptide in accordance with thisaspect of the present invention.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents; such methods are well know to those skilled in the art.

Plant Transformation Methods:

Isolated nucleic acid acids of the present invention can be introducedinto plants according to techniques known in the art. Generally,recombinant expression cassettes as described above and suitable fortransformation of plant cells are prepared. Techniques for transforminga wide variety of higher plant species are well known and described inthe technical, scientific, and patent literature. See, for example,Weising, et al., (1988) Ann. Rev. Genet. 22:421-477. For example, theDNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation, PEG poration,particle bombardment, silicon fiber delivery, or microinjection of plantcell protoplasts or embryogenic callus. Alternatively, the DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria. See, U.S. Pat. No.5,591,616.

The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski, et al., (1984) Embo J.3:2717-2722. Electroporation techniques are described in Fromm, et al.,(1985) Proc. Natl. Acad. Sci. (USA) 82:5824. Ballistic transformationtechniques are described in Klein, et al., (1987) Nature 327:70-73 andby Tomes, D., et al., (1995) Plant Cell, Tissue and Organ Culture:Fundamental Methods, Eds. O. L. Gamborg and G. C. Phillips, Chapter 8,pgs. 197-213. (See also, Tomes, et al., U.S. Pat. Nos. 5,886,244;6,258,999; 6,570,067; 5,879,918)

Agrobacterium tumefaciens-meditated transformation techniques are welldescribed in the scientific literature. See, for example Horsch, et al.,(1984) Science 233:496-498, and Fraley, et al., (1983) Proc. Natl. Acad.Sci_(USA) 80:4803. Although Agrobacterium is useful primarily in dicots,certain monocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller, Genetic Engineering, vol. 6, P W J Rigby, Ed.,London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J.,DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985),Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:1353),(3) the vortexing method (see, e.g., Kindle, (1990) Proc. Nat'l. Acad.Sci. (USA) 87:1228.

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou, et al., (1983) Methods in Enzymology,101:433; D. Hess, (1987) Intern. Rev. Cytol., 107:367; Luo, et al.,(1988) Plant Mol. Biol. Reporter, 6:165. Expression of polypeptidecoding genes can be obtained by injection of the DNA into reproductiveorgans of a plant as described by Pena, et al., (1987) Nature 325:274.DNA can also be injected directly into the cells of immature embryos andthe rehydration of desiccated embryos as described by Neuhaus, et al.,(1987) Theor. Appl. Genet., 75:30; and Benbrook, et al., (1986)Proceedings Bio Expo., Butterworth, Stoneham, Mass., pp. 27-54. Avariety of plant viruses that can be employed as vectors are known inthe art and include cauliflower mosaic virus (CaMV), geminivirus, bromemosaic virus, and tobacco mosaic virus.

Regeneration of Transformed Plants

Transformed plant cells that are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, forexample, U.S. Pat. No. 5,736,369.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans, et al.,(1983) Protoplasts Isolation and Culture, Handbook of Plant CellCulture, Macmillilan Publishing Company, New York, pp. 124-176; andBinding, (1985) Regeneration of Plants, Plant Protoplasts, CRC Press,Boca Raton, pp. 21-73.

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horsch,et al., (1985) Science 227:1229-1231. In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed as described by Fraley, et al., (1983) Proc. Nat'l. Acad.Sci. (U.S.A) 80:4803. This procedure typically produces shoots withintwo to four weeks and these transformant shoots are then transferred toan appropriate root-inducing medium containing the selective agent andan antibiotic to prevent bacterial growth. Transgenic plants of thepresent invention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee, et al., (1987) Ann. Rev. of Plant Phys. 38:467-486. Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3^(rd) edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed-propagated crops, mature transgenic plants canbe self-crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype. Mature transgenic plants can also be crossed withother appropriate plants, generally another inbred or hybrid, including,for example, an isogenic untransformed inbred.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these plants comprise the introduced nucleic acidsequences.

Transgenic plants expressing the selectable marker can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then be analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

Some embodiments comprise a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous (aka hemizygous)transgenic plant that contains a single added heterologous nucleic acid,germinating some of the seed produced and analyzing the resulting plantsproduced for altered expression of a polynucleotide of the presentinvention relative to a control plant (i.e., native, non-transgenic).Back-crossing to a parental plant and out-crossing with a non-transgenicplant, or with a plant transgenic for the same or another trait ortraits, are also contemplated.

It is also expected that the transformed plants will be used intraditional breeding programs, including TOPCROSS pollination systems asdisclosed in U.S. Pat. No. 5,706,603 and U.S. Pat. No. 5,704,160, thedisclosure of each of which is incorporated herein by reference.

Polynucleotide Assays

This invention is also related to the use of the cytokinin biosyntheticenzyme polynucleotides in markers to assist in a breeding program, asdescribed for example in PCT publication US89/00709. The DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki, et al., (1986) Nature 324:163-166) prior to analysis. RNA orcDNA may also be used in the same ways. As an example, PCR primerscomplementary to the nucleic acid encoding the cytokinin biosyntheticenzymes can be used to identify and analyze cytokinin biosyntheticenzyme presence and expression. Using PCR, characterization of the genepresent in a particular tissue or plant variety may be made by ananalysis of the genotype of the tissue or variety. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the genotype of a reference sequence.Point mutations can be identified by hybridizing amplified DNA toradiolabeled cytokinin biosynthetic enzyme RNA or alternatively,radiolabeled cytokinin biosynthetic enzyme antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent tags.

Genetic typing of various varieties of plants based on DNA sequencedifferences may be achieved by detection of alteration inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents. Small sequence deletions and insertions can bevisualized by high resolution gel electrophoresis. DNA fragments ofdifferent sequences may be distinguished on denaturing formamidegradient gels in which the mobilities of different DNA fragments areretarded in the gel at different positions according to their specificmelting or partial melting temperatures (see, e.g., Myers, et al.,(1985) Science, 230:1242).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton, et al., (1985) Proc. Nat'l. Acad. Sci.(USA) 85:4397-4401).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”)) and Southernblotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

A mutation may be ascertained, for example, by a DNA sequencing assay.Samples are processed by methods known in the art to capture the RNA.First strand cDNA is synthesized from the RNA samples by adding anoligonucleotide primer consisting of sequences that hybridize to aregion on the mRNA. Reverse transcriptase and deoxynucleotides are addedto allow synthesis of the first strand cDNA. Primer sequences aresynthesized based on the DNA sequences of the cytokinin modulatingenzymes of the invention. The primer sequence is generally comprised ofat least 15 consecutive bases, and may contain at least 30 or even 50consecutive bases.

Cells carrying mutations or polymorphisms in the gene of the presentinvention may also be detected at the DNA level by a variety oftechniques. The DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki, et al., (1986) Nature324:163-166) prior to analysis. RT-PCR can also be used to detectmutations. It is particularly preferred to use RT-PCR in conjunctionwith automated detection systems, such as, for example, GeneScan. RNA orcDNA may also be used for the same purpose, PCR or RT-PCR. As anexample, PCR primers complementary to the nucleic acid encodingcytokinin biosynthetic enzymes can be used to identify and analyzemutations. Examples of representative primers are shown below. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to radiolabeledRNA, or alternatively, radiolabeled antisense DNA sequences. Whileperfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures,preferably point mutations are identified by sequence analysis. Primersused for detection of mutations or polymorphisms in the ipt gene:

(SEQ ID NO: 40) 5′GCGTCCAATGCTGTCCTCAACTA3′ (SEQ ID NO: 41)5′GCTCTCCTCGTCTGCTAACTCGT3′

The above primers may be used for amplifying cytokinin biosyntheticenzyme cDNA or genomic clones isolated from a sample derived from anindividual plant. The invention also provides the primers above with 1,2, 3 or 4 nucleotides removed from the 5′ and/or the 3′ end. The primersmay be used to amplify the gene isolated from the individual such thatthe gene may then be subject to various techniques for elucidation ofthe DNA sequence. In this way, mutations in the DNA sequence may beidentified.

Polypeptide Assays

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of cytokininbiosynthetic enzymes in cells and tissues, including determination ofnormal and abnormal levels. Thus, for instance, a diagnostic assay inaccordance with the invention for detecting expression of cytokininbiosynthetic enzymes compared to normal control tissue samples may beused to detect unacceptable levels of expression. Assay techniques thatcan be used to determine levels of polypeptides of the present inventionin a sample derived from a plant source are well-known to those of skillin the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these, ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to the polypeptide, preferablya monoclonal antibody. In addition, a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as a radioactive, fluorescentor enzymatic reagent, in this example horseradish peroxidase enzyme.

To carry out an ELISA, a sample is removed from a host and incubated ona solid support, e.g., a polystyrene dish, which binds the proteins inthe sample. Any free protein binding sites on the dish are then coveredby incubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish, during whichtime the monoclonal antibodies attach to any cytokinin biosyntheticenzymes attached to the polystyrene dish. Unbound monoclonal antibody iswashed out with buffer. The reporter antibody linked to horseradishperoxidase is placed in the dish, resulting in binding of the reporterantibody to any monoclonal antibody bound to cytokinin biosyntheticenzyme. Unattached reporter antibody is then washed out. Reagents forperoxidase activity, including a calorimetric substrate, are then addedto the dish. Immobilized peroxidase, linked to cytokinin biosyntheticenzyme through the primary and secondary antibodies, produces a coloredreaction product. The amount of color developed in a given time periodindicates the amount of cytokinin biosynthetic enzyme present in thesample. Quantitative results typically are obtained by reference to astandard curve.

A competition assay may be employed wherein antibodies specific tocytokinin biosynthetic enzymes are attached to a solid support, andlabeled enzyme derived from the host is passed over the solid support.The amount of label detected attached to the solid support can becorrelated to a quantity of cytokinin biosynthetic enzyme in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as immunogens to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal, or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptide can be used to generate antibodiesbinding the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique that providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,(1975) Nature 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor, et al., (1983) Immunology Today 4:72) andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., (1985) pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc.).

Hybridoma cell lines secreting the monoclonal antibody are anotheraspect of this invention.

Techniques described for the production of single-chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single-chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

The above-described antibodies may be employed to isolate or identifyclones expressing the polypeptide or to purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Polypeptide derivatives include antigenically or immunologicallyequivalent derivatives that form a particular aspect of this invention.

The term ‘antigenically equivalent derivative’ as used hereinencompasses a polypeptide or its equivalent which will be specificallyrecognized by certain antibodies which, when raised to the protein orpolypeptide according to the present invention, interfere with theimmediate physical interaction between the antibody and its cognateantigen.

The term “immunologically equivalent derivative” as used hereinencompasses a peptide or its equivalent which, when used in a suitableformulation to raise antibodies in a vertebrate, results in antibodieswhich act to interfere with the immediate physical interaction betweenthe antibody and its cognate antigen.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof, is used as an antigen toimmunize a mouse or other animal, such as a rat, guinea pig, goat,rabbit, sheep, bovine or chicken. The fusion protein may providestability to the polypeptide. The antigen may be associated, for exampleby conjugation, with an immunogenic carrier protein, for example bovineserum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively amultiple antigenic peptide comprising multiple copies of the protein orpolypeptide, or an antigenically or immunologically equivalentpolypeptide thereof, may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Alternatively, phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty,J., et al., (1990), Nature 348:552-554; Marks, J., et al., (1992)Biotechnology 10:779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T., et al., (1991) Nature352:624-628).

The antibody should be screened again for high affinity to thepolypeptide and/or fusion protein.

As mentioned above, a fragment of the final antibody may be prepared.

The antibody may be either intact antibody of Mr approximately 150,000or a derivative of it, for example a Fab fragment or a Fv fragment asdescribed in Sierra, A and Pluckthun, A., (1988) Science 240:1038-1040.If two antigen binding domains are present, each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The antibody of the invention, as mentioned above, may be prepared byconventional means, for example by established monoclonal antibodytechnology (Kohler, G. and Milstein, C., (1975) Nature 256:495-497) orusing recombinant means e.g. combinatorial libraries, for example asdescribed in Huse, W. D., et al., (1989) Science 246:1275-1281.

Preferably the antibody is prepared by expression of a DNA polymerencoding said antibody in an appropriate expression system such asdescribed above for the expression of polypeptides of the invention. Thechoice of vector for the expression system will be determined in part bythe host, which may be a prokaryotic cell, such as E. coli (preferablystrain B) or Streptomyces sp. or a eukaryotic cell, such as a mouseC127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous orunicellular fungi or insect cell. The host may also be a transgenicanimal or a transgenic plant for example as described in Hiatt, A., etal., (1989) Nature 340:76-78. Suitable vectors include plasmids,bacteriophages, cosmids and recombinant viruses, derived from, forexample, baculoviruses and vaccinia.

The Fab fragment may also be prepared from its parent monoclonalantibody by enzyme treatment, for example using papain to cleave the Fabportion from the Fc portion.

Cytokinin Biosynthetic Enzyme Binding Molecules and Assays

This invention also provides a method for identification of molecules,such as binding molecules, that bind the cytokinin biosynthetic enzymes.Genes encoding proteins that bind the enzymes, such as binding proteins,can be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting. Such methods aredescribed in many laboratory manuals such as, for instance, Coligan, etal., (1991) Current Protocols in Immunology 1(2) Chapter 5.

For instance, expression cloning may be employed for this purpose. Tothis end, polyadenylated RNA is prepared from a cell expressing thecytokinin biosynthetic enzymes, a cDNA library is created from this RNA,the library is divided into pools, and the pools are transfectedindividually into cells that are not expressing the enzyme. Thetransfected cells then are exposed to labeled enzyme. The enzyme can belabeled by a variety of well-known techniques, including standardmethods of radio-iodination or inclusion of a recognition site for asite-specific protein kinase. Following exposure, the cells are fixedand binding of enzyme is determined. These procedures conveniently arecarried out on glass slides.

Pools are identified of cDNA that produced cytokinin biosyntheticenzyme-binding cells. Sub-pools are prepared from these positives,transfected into host cells and screened as described above. Using aniterative sub-pooling and re-screening process, one or more singleclones that encode the putative binding molecule can be isolated.

Alternatively, a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a binding molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis(“PAGE”) and exposed to X-ray film. The labeled complex containing theligand-binding can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative binding molecule.

Polypeptides of the invention also can be used to assess cytokininbiosynthetic enzyme binding capacity of cytokinin biosynthetic enzymebinding molecules, such as binding molecules, in cells or in cell-freepreparations.

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics.

Anti-cytokinin biosynthetic enzyme antibodies represent a useful classof binding molecules contemplated by this invention.

Antagonists and Agonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose that enhance or block the action of cytokinin biosynthetic enzymeson cells, such as interaction with substrate molecules. An antagonist isa compound that decreases the natural biological functions of theenzymes. A particular enzyme to be targeted in this regard is cytokininoxidase.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to cytokinin oxidase and therebyinhibit or extinguish its activity. Potential antagonists also may besmall organic molecules, a peptide, a polypeptide such as a closelyrelated protein or antibody, that binds the same sites on a bindingmolecule, such as a cytokinin oxidase binding molecule, without inducingcytokinin metabolic enzyme-induced activities, thereby preventing theaction of the enzyme by excluding the enzyme from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such as binding molecules, such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small organic molecules, peptides orpeptide-like molecules.

Other potential antagonists include molecules that affect the expressionof the gene encoding cytokinin biosynthetic enzymes (e.g.transactivation inhibitors). Other potential antagonists includeantisense molecules. Antisense technology can be used to control geneexpression through antisense DNA or RNA or through double- ortriple-helix formation. Antisense techniques are discussed, for example,in—Okano, J., (1991) Neurochem. 56:560; OLIGODEOXYNUCLEOTIDES ASANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance Lee, etal., (1979) Nucleic Acids Research 6:3073; Cooney, et al., (1988)Science 241:456 (1988); and Dervan, et al., (1991) Science 251:1360. Themethods are based on binding of a polynucleotide to a complementary DNAor RNA. For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription, thereby preventingtranscription and the production of cytokinin biosynthetic enzymes. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into cytokinin biosynthetic enzymes.The oligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of cytokinin biosynthetic enzymes.

The DNAs of this invention may also be employed to co-suppress orsilence the cytokinin metabolic enzyme genes; for example, as describedin PCT Patent Application Publication WO 98/36083.

The antagonists may be employed for instance to increase the levels ofcytokinin and/or decrease the available auxin in plant cells.

Alternatively, this invention provides methods for screening foragonists, those molecules that act to increase the natural biologicalfunction of enzymes. Targets in this regard include enzymes such ipt,β-glucosidase, and iaa-1.

Potential agonists include small organic molecules, peptides,polypeptides and antibodies that bind to a biosynthetic enzyme andthereby stimulate or increase its activity. Potential agonists also maybe small organic molecules, a peptide, a polypeptide such as a closelyrelated protein or antibody that binds to sites on a binding molecule,such as a ipt binding molecule and promotes cytokinin metabolicenzyme-induced activities, thereby enhancing the action of the enzyme.

Potential agonists include small molecules that bind to and occupy theallosteric sites of the enzyme thereby promoting binding to cellularbinding molecules, such as substrates, such that normal biologicalactivity is enhanced. Examples of small molecules include but are notlimited to small organic molecules, peptides or peptide-like molecules.

Other potential agonists include molecules that affect the expression ofthe gene encoding cytokinin biosynthetic enzymes (e.g.,transactivatiors).

“Stacking” of Constructs and Traits

In certain embodiments the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The polynucleotides of the present invention may bestacked with any gene or combination of genes, and the combinationsgenerated can include multiple copies of any one or more of thepolynucleotides of interest. The desired combination may affect one ormore traits; that is, certain combinations may be created for modulationof gene expression affecting cytokinin activity. For example,up-regulation of cytokinin synthesis may be combined withdown-regulation of cytokinin oxidase expression. Other combinations maybe designed to produce plants with a variety of desired traits,including but not limited to traits desirable for animal feed such ashigh oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids(e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802;and 5,703,409); barley high lysine (Williamson, et al., (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122); and high methionine proteins(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al.,(1988) Gene 71:359; and Musumura, et al., (1989) Plant Mol. Biol.12:123)); increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)), thedisclosures of which are herein incorporated by reference. Thepolynucleotides of the present invention can also be stacked with traitsdesirable for insect, disease or herbicide resistance (e.g., Bacillusthuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene 48:109);lectins (Van Damme, et al., (1994) Plant Mol. Biol. 24:825); fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones, et al., (1994) Science 266:789; Martin, et al.,(1993) Science 262:1432; Mindrinos, et al., (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene)); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE) and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert, et al., (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)),the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the present invention withpolynucleotides affecting agronomic traits such as male sterility (e.g.,see U.S. Pat. No. 5,583,210), stalk strength, flowering time, ortransformation technology traits such as cell cycle regulation or genetargeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821), the disclosuresof which are herein incorporated by reference.

These stacked combinations can be created by any method, including butnot limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences of interest can be driven bythe same promoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of a polynucleotide of interest. This may be accompanied byany combination of other suppression cassettes or over-expressioncassettes to generate the desired combination of traits in the plant.

Use in Breeding Methods

The transformed plants of the invention may be used in a plant breedingprogram. The goal of plant breeding is to combine, in a single varietyor hybrid, various desirable traits. For field crops, these traits mayinclude, for example, resistance to diseases and insects, tolerance toheat and drought, reduced time to crop maturity, greater yield, andbetter agronomic quality. With mechanical harvesting of many crops,uniformity of plant characteristics such as germination and standestablishment, growth rate, maturity, and plant and ear height, isdesirable. Traditional plant breeding is an important tool in developingnew and improved commercial crops. This invention encompasses methodsfor producing a maize plant by crossing a first parent maize plant witha second parent maize plant wherein one or both of the parent maizeplants is a transformed plant displaying enhanced vigor, as describedherein.

Plant breeding techniques known in the art and used in a maize plantbreeding program include, but are not limited to, recurrent selection,bulk selection, mass selection, backcrossing, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, doubled haploids, andtransformation. Often combinations of these techniques are used.

The development of maize hybrids in a maize plant breeding programrequires, in general, the development of homozygous inbred lines, thecrossing of these lines, and the evaluation of the crosses. There aremany analytical methods available to evaluate the result of a cross. Theoldest and most traditional method of analysis is the observation ofphenotypic traits. Alternatively, the genotype of a plant can beexamined.

A genetic trait which has been engineered into a particular maize plantusing transformation techniques, could be moved into another line usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach is commonly used tomove a transgene from a transformed maize plant to an elite inbred line,and the resulting progeny would then comprise the transgene(s). Also, ifan inbred line was used for the transformation then the transgenicplants could be crossed to a different inbred in order to produce atransgenic hybrid maize plant. As used herein, “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, while different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrids. During theinbreeding process in maize, the vigor of the lines decreases. Vigor isrestored when two different inbred lines are crossed to produce thehybrid. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid created by crossing a defined pairof inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

Transgenic plants of the present invention may be used to produce asingle cross hybrid, a three-way hybrid or a double cross hybrid. Asingle cross hybrid is produced when two inbred lines are crossed toproduce the F1 progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F1 hybridsare crossed again (A×B)×(C×D). A three-way cross hybrid is produced fromthree inbred lines where two of the inbred lines are crossed (A×B) andthen the resulting F1 hybrid is crossed with the third inbred (A×B)×C.Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lostin the next generation (F2). Consequently, seed produced by hybrids isconsumed rather than planted.

In accordance with the invention, nucleotide sequences are provided thatallow initiation of transcription in seed. The sequences of theinvention comprise transcriptional initiation regions associated withseed formation and seed tissues. Thus, the compositions of the presentinvention comprise novel nucleotide sequences for regulatory sequences.

A method for expressing an isolated nucleotide sequence in a plant usingthe transcriptional initiation sequences disclosed herein is provided.Suitable techniques are described by Maniatis, T., Fritsch, E. F. andSambrook, J. in MOLECULAR CLONING, A Laboratory Manual (2nd edition 1989Cold Spring Harbor Laboratory). The method comprises transforming aplant cell with a transformation vector that comprises an isolatednucleotide sequence operably linked to the promoter of the presentinvention and regenerating a stably transformed plant from thetransformed plant cell. In this manner, the promoter is useful forcontrolling the expression of endogenous as well as exogenous productsin a seed-preferred manner.

Under the transcriptional initiation regulation of the seed-preferredpromoter region will be a sequence of interest, which will provide formodification of the phenotype of the seed. Such modification includesmodulating the production of an endogenous product, as to amount,relative distribution, or the like, or production of an exogenousexpression product to provide for a novel function or product in theseed.

By “seed-preferred” is intended favored expression in the seed,including at least one of embryo, kernel, pericarp, endosperm, nucellus,aleurone, pedicel, and the like.

By “regulatory element” is intended sequences responsible fortissue-preferred and temporally-preferred expression of the associatedcoding sequence, including promoters, terminators, enhancers, introns,and the like.

By “promoter” is intended a regulatory region of DNA usually comprisinga TATA box capable of directing RNA polymerase II to initiate RNAsynthesis at the appropriate transcription initiation site for aparticular coding sequence. A promoter can additionally comprise otherrecognition sequences generally positioned upstream or 5′ to the TATAbox, referred to as upstream promoter elements, which influence thetranscription initiation rate. It is recognized that having identifiedthe nucleotide sequences for the promoter region disclosed herein, it iswithin the state of the art to isolate and identify further regulatoryelements in the 5′ untranslated region upstream from the particularpromoter region identified herein. Thus the promoter region disclosedherein is generally further defined by comprising upstream regulatoryelements such as those responsible for tissue-preferred andtemporally-preferred expression of the coding sequence, enhancers, andthe like. In the same manner, the promoter elements that enableexpression in the desired tissue such as the seed can be identified,isolated, and used with other core promoters to confirm seed-preferredexpression.

The isolated promoter sequences of the present invention can be modifiedto provide for a range of expression levels of the isolated nucleotidesequence. Less than the entire promoter region can be utilized and theability to drive seed-preferred expression retained. However, it isrecognized that expression levels of mRNA can be decreased withdeletions of portions of the promoter sequence. Thus, the promoter canbe modified to be a weak or strong promoter. Generally, by “weakpromoter” is intended a promoter that drives expression of a codingsequence at a low level. By “low level” is intended levels of about1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts. Generally, at least about 20nucleotides of an isolated promoter sequence will be used to driveexpression of a nucleotide sequence.

It is recognized that to increase transcription levels, enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

A promoter of the present invention can be isolated from the 5′untranslated region flanking the transcription initiation site of itsrespective coding sequence. Likewise, the terminator can be isolatedfrom the 3′ untranslated region flanking the stop codon of itsrespective coding sequence.

The term “isolated” refers to material, such as a nucleic acid orprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment or (2) if the material is in its naturalenvironment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art. A sequence for the promoter regioneep1 is set forth in SEQ ID NO: 7. A sequence for the promoter regioneep2 is set forth in SEQ ID NO: 18.

The eep1 promoter set forth in SEQ ID NO: 7 is 960 nucleotides in lengthA putative CAAT motif is found 308 bp upstream of the start oftranslation and a putative TATA motif is found 139 bp upstream form thestart of translation. The promoter was isolated from EST sequences foundin maize tissue libraries of 4 and 6 DAP embryo sacs, as well as 5 and 7DAP whole kernels. The eep1 promoter can address expression problems byproviding expression in seed tissues during early stages of seeddevelopment.

The eep2 promoter set forth in SEQ ID NO: 18 is 1027 nucleotides inlength. The promoter was isolated from an EST sequence found in maizetissue libraries of 4 DAP (days after pollination) embryo sacs and ishighly specific for early kernel and endosperm expression, as determinedby EST distribution among libraries and by Lynx MPSS profiling.

The promoter regions of the invention may be isolated from any plant,including, but not limited to, maize (corn; Zea mays), canola (Brassicanapus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats, barley, vegetables, ornamentals, and conifers.Preferably, plants include maize, soybean, sunflower, safflower, canola,wheat, barley, rye, alfalfa, and sorghum.

Promoter sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the promotersequences set forth herein. In these techniques, all or part of theknown promoter sequence is used as a probe which selectively hybridizesto other sequences present in a population of cloned genomic DNAfragments (i.e. genomic libraries) from a chosen organism. Methods thatare readily available in the art for the hybridization of nucleic acidsequences may be used to obtain sequences which correspond to thepromoter of the present invention.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g., Innis, et al. (1990) PCR Protocols, AGuide to Methods and Applications, eds., Academic Press).

In general, sequences that correspond to the promoter sequence of thepresent invention and hybridize to the promoter sequence disclosedherein will be at least 50% homologous, 55% homologous, 60% homologous,65% homologous, 70% homologous, 75% homologous, 80% homologous, 85%homologous, 90% homologous, 95% homologous and even 98% homologous ormore with the disclosed sequence.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “percentage of sequenceidentity”, and (d) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, a segment of afull-length promoter sequence, or the complete promoter sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length and optionally can be30, 40, 50, 100, or more contiguous nucleotides in length. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

(c) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

(d) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters.

Methods of aligning sequences for comparison are well known in the art.Gene comparisons can be determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol.215:403-410; see also, www.ncbi.nlm.nih.gov/BLAST/) searches underdefault parameters for identity to sequences contained in the BLAST“GENEMBL” database. A sequence can be analyzed for identity to allpublicly available DNA sequences contained in the GENEMBL database usingthe BLASTN algorithm under the default parameters. Identity to thesequence of the present invention would mean a polynucleotide sequencehaving at least 65% sequence identity, more preferably at least 70%sequence identity, more preferably at least 75% sequence identity, morepreferably at least 80% identity, more preferably at least 85% sequenceidentity, more preferably at least 90% sequence identity and mostpreferably at least 95% sequence identity wherein the percent sequenceidentity is based on the entire promoter region.

For purposes of defining the present invention, GAP (Global AlignmentProgram) is used. GAP uses the algorithm of Needleman and Wunsch (1970)J. Mol. Biol. 48:443-453, to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps. GAP considers all possible alignments and gap positions andcreates the alignment with the largest number of matched bases and thefewest gaps. It allows for the provision of a gap creation penalty and agap extension penalty in units of matched bases. GAP must make a profitof gap creation penalty number of matches for each gap it inserts. If agap extension penalty greater than zero is chosen, GAP must, inaddition, make a profit for each gap inserted of the length of the gaptimes the gap extension penalty. Default gap creation penalty values andgap extension penalty values in Version 10 of the Wisconsin Package®(Accelrys, Inc., San Diego, Calif.) for protein sequences are 8 and 2,respectively. For nucleotide sequences the default gap creation penaltyis 50 while the default gap extension penalty is 3. The gap creation andgap extension penalties can be expressed as an integer selected from thegroup of integers consisting of from 0 to 200. Thus, for example, thegap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Package® (Accelrys,Inc., San Diego, Calif.) is BLOSUM62 (see, Henikoff & Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915).

Sequence fragments with high percent identity to the sequences of thepresent invention also refer to those fragments of a particular promotersequence disclosed herein that operate to promote the seed-preferredexpression of an operably-linked isolated nucleotide sequence. Thesefragments will comprise at least about 20 contiguous nucleotides,preferably at least about 50 contiguous nucleotides, more preferably atleast about 75 contiguous nucleotides, even more preferably at leastabout 100 contiguous nucleotides of the particular promoter nucleotidesequence disclosed herein. The nucleotides of such fragments willusually comprise the TATA recognition sequence of the particularpromoter sequence. Such fragments can be obtained by use of restrictionenzymes to cleave the naturally occurring promoter sequences disclosedherein; by synthesizing a nucleotide sequence from thenaturally-occurring DNA sequence; or through the use of PCR technology.See particularly, Mullis, et al., (1987) Methods Enzymol. 155:335-350,and Erlich, ed. (1989) PCR Technology (Stockton Press, New York). Again,variants of these fragments, such as those resulting from site-directedmutagenesis, are encompassed by the compositions of the presentinvention.

Nucleotide sequences comprising at least about 20 contiguous sequencesof the sequence set forth in SEQ ID NO: 10 are encompassed. Thesesequences can be isolated by hybridization, PCR, and the like. Suchsequences encompass fragments capable of driving seed-preferredexpression, fragments useful as probes to identify similar sequences, aswell as elements responsible for temporal or tissue specificity.

Biologically active variants of the promoter sequence are alsoencompassed by the compositions of the present invention. A regulatory“variant” is a modified form of a promoter wherein one or more baseshave been modified, removed or added. For example, a routine way toremove part of a DNA sequence is to use an exonuclease in combinationwith DNA amplification to produce unidirectional nested deletions ofdouble-stranded DNA clones. A commercial kit for this purpose is soldunder the trade name Exo-Size™ (New England Biolabs, Beverly, Mass.).Briefly, this procedure entails incubating exonuclease III with DNA toprogressively remove nucleotides in the 3′ to 5′ direction at 5′overhangs, blunt ends or nicks in the DNA template. However, exonucleaseIII is unable to remove nucleotides at 3′, 4-base overhangs. Timeddigests of a clone with this enzyme produce unidirectional nesteddeletions.

One example of a regulatory sequence variant is a promoter formed bycausing one or more deletions in a larger promoter. The 5′ portion of apromoter up to the TATA box near the transcription start site can bedeleted without abolishing promoter activity, as described by Zhu. etal., (1985) The Plant Cell 7:1681-89. Such variants should retainpromoter activity, particularly the ability to drive expression in seedor seed tissues. Biologically active variants include, for example, thenative regulatory sequences of the invention having one or morenucleotide substitutions, deletions or insertions. Activity can bemeasured by Northern blot analysis, reporter activity measurements whenusing transcriptional fusions, and the like. See, for example, Sambrook,et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.), herein incorporatedby reference.

The nucleotide sequences for the seed-preferred promoter disclosed inthe present invention, as well as variants and fragments thereof, areuseful in the genetic manipulation of any plant when operably linkedwith an isolated nucleotide sequence whose expression is to becontrolled to achieve a desired phenotypic response. By “operablylinked” is intended that the transcription or translation of theisolated nucleotide sequence is under the influence of the regulatorysequence. In this manner, a nucleotide sequence for the promoter of theinvention may be provided in an expression cassette along with anisolated nucleotide sequence for expression in the plant of interest,more particularly in the seed of the plant. Such an expression cassetteis provided with a plurality of restriction sites for insertion of thenucleotide sequence to be under the transcriptional control of thepromoter.

The genes of interest expressed under the direction of the promoter ofthe invention can be used for varying the phenotype of seeds. This canbe achieved by increasing expression of endogenous or exogenous productsin seeds. Alternatively, results can be achieved by providing for areduction of expression of one or more endogenous products, particularlyenzymes or cofactors in the seed. These modifications result in a changein phenotype of the transformed seed. It is recognized that the promotermay be used with its native coding sequence to increase or decreaseexpression, resulting in a change in phenotype in the transformed seed.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas Zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, and grain characteristics. Still other categories oftransgenes include genes for inducing expression of exogenous productssuch as enzymes, cofactors, and hormones from plants and othereukaryotes as well as prokaryotic organisms. It is recognized that anygene of interest, including the native coding sequence, can be operablylinked to the regulatory elements of the invention and expressed in theseed.

Modifications that affect grain traits include increasing the content ofoleic acid, or altering levels of saturated and unsaturated fatty acids.Likewise, increasing the levels of lysine and sulfur-containing aminoacids may be desired as well as the modification of starch type andcontent in the seed. Hordothionin protein modifications are described inWO 9416078 filed Apr. 10, 1997; WO 9638562 filed Mar. 26, 1997; WO9638563 filed Mar. 26, 1997 and U.S. Pat. No. 5,703,409 issued Dec. 30,1997; the disclosures of which are incorporated herein by reference.Another example is lysine and/or sulfur-rich seed protein encoded by thesoybean 2S albumin described in WO 9735023 filed Mar. 20, 1996, and thechymotrypsin inhibitor from barley, Williamson, et al., (1987) Eur. J.Biochem. 165:99-106, the disclosures of each are incorporated byreference.

Derivatives of the following genes can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL), is derived from barley chymotrypsin inhibitor,WO 9820133 filed Nov. 1, 1996 the disclosure of which is incorporatedherein by reference. Other proteins include methionine-rich plantproteins such as from sunflower seed, Lilley, et al., (1989) Proceedingsof the World Congress on Vegetable Protein Utilization in Human Foodsand Animal Feedstuffs; Applewhite, H. (ed.); American Oil Chemists Soc.,Champaign, Ill. 497-502, incorporated herein by reference; corn,Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al.,(1988) Gene 71:359, both incorporated herein by reference; and rice,Musumura, et al., (1989) Plant Mol. Biol. 12:123, incorporated herein byreference. Other important genes encode glucans, Floury 2, growthfactors, seed storage factors and transcription factors.

Agronomic traits in seeds can be improved by altering expression ofgenes that: affect the response of seed growth and development duringenvironmental stress, Cheikh-N, et al., (1994) Plant Physiol.106(1):45-51 and genes controlling carbohydrate metabolism to reducekernel abortion in maize, Zinselmeier, et al., (1995) Plant Physiol.107(2):385-391. These include, for example, genes encoding cytokininbiosynthesis enzymes, such as isopentenyl transferase; genes encodingcytokinin catabolic enzymes, such as cytokinin oxidase; genes encodingpolypeptides involved in regulation of the cell cycle, such as CyclinDor cdc25; genes encoding cytokinin receptors or sensors, such as CRE1,CKI1, and CKI2, histidine phospho-transmitters, or cytokinin responseregulators.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example: Bacillus thuringiensis endotoxin genes,U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881;Geiser, et al., (1986) Gene 48:109; lectins, Van Damme, et al., (1994)Plant Mol. Biol. 24:825; and the like.

Genes encoding disease resistance traits include: detoxification genes,such as against fumonosin (WO 9606175 filed Jun. 7, 1995); avirulence(avr) and disease resistance (R) genes, Jones, et al., (1994) Science266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al.,(1994) Cell 78:1089; and the like.

Commercial traits can also be encoded on a gene(s) which could alter orincrease for example, starch for the production of paper, textiles andethanol, or provide expression of proteins with other commercial uses.Another important commercial use of transformed plants is the productionof polymers and bioplastics such as described in U.S. Pat. No. 5,602,321issued Feb. 11, 1997. Genes such as B-Ketothiolase, PHBase(polyhydroxyburyrate synthase) and acetoacetyl-CoA reductase (see,Schubert, et al., (1988) J. Bacteriol 170(12):5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof seed proteins, particularly modified seed proteins having improvedamino acid distribution to improve the nutrient value of the seed can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. By“antisense DNA nucleotide sequence” is intended a sequence that is ininverse orientation to the 5′-to-3′ normal orientation of thatnucleotide sequence. When delivered into a plant cell, expression of theantisense DNA sequence prevents normal expression of the DNA nucleotidesequence for the targeted gene. The antisense nucleotide sequenceencodes an RNA transcript that is complementary to and capable ofhybridizing with the endogenous messenger RNA (mRNA) produced bytranscription of the DNA nucleotide sequence for the targeted gene. Inthis case, production of the native protein encoded by the targeted geneis inhibited to achieve a desired phenotypic response. Thus theregulatory sequences disclosed herein can be operably linked toantisense DNA sequences to reduce or inhibit expression of a nativeprotein in the plant seed.

The expression cassette will also include, at the 3′ terminus of theisolated nucleotide sequence of interest, a transcriptional andtranslational termination region functional in plants. The terminationregion can be native with the promoter nucleotide sequence of thepresent invention, can be native with the DNA sequence of interest, orcan be derived from another source.

Other convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903; Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample: EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein, et al., (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus),Allison, et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus),Virology 154:9-20; human immunoglobulin heavy-chain binding protein(BiP), Macejak, et al., (1991) Nature 353:90-94; untranslated leaderfrom the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling,et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV),Gallie, et al., (1989) Molecular Biology of RNA, pages 237-256; andmaize chlorotic mottle virus leader (MCMV) Lommel, et al., (1991)Virology 81:382-385. See also, Della-Cioppa, et al., (1987) PlantPhysiology 84:965-968. The cassette can also contain sequences thatenhance translation and/or mRNA stability such as introns.

In those instances where it is desirable to have the expressed productof the isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleicacids). Vectors and procedures for cloning and expression in E. coli arediscussed in Sambrook, et al. (supra).

The transformation vector, comprising the promoter of the presentinvention operably linked to an isolated nucleotide sequence in anexpression cassette, can also contain at least one additional nucleotidesequence for a gene to be co-transformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

Vectors that are functional in plants can be binary plasmids derivedfrom Agrobacterium. Such vectors are capable of transforming plantcells. These vectors contain left and right border sequences that arerequired for integration into the host (plant) chromosome. At minimum,between these border sequences is the gene to be expressed under controlof the regulatory elements of the present invention. In one embodiment,a selectable marker and a reporter gene are also included. For ease ofobtaining sufficient quantities of vector, a bacterial origin thatallows replication in E. Coli can be used.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample: Jefferson, et al., (1991) in Plant Molecular Biology Manual,ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, etal., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO J.9:2517-2522; Kain, et al., (1995) BioTechniques 19:650-655; and Chiu, etal., (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to:genes encoding resistance to chloramphenicol, Herrera Estrella, et al.,(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella, et al., (1983)Nature 303:209-213; Meijer, et al., (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron, et al., (1985) Plant Mol. Biol. 5:103-108; Zhijian,et al., (1995) Plant Science 108:219-227; streptomycin, Jones, et al.,(1987) Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard, etal., (1996) Transgenic Res. 5:131-137; bleomycin, Hille, et al., (1990)Plant Mol. Biol. 7:171-176; sulfonamide, Guerineau, et al., (1990) PlantMol. Biol. 15:127-136; bromoxynil, Stalker, et al., (1988) Science242:419-423; glyphosate, Shaw, et al., (1986) Science 233:478-481;phosphinothricin, DeBlock, et al., (1987) EMBO J. 6:2513-2518.

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to: GUS (β-glucoronidase), Jefferson (1987) Plant Mol.Biol. Rep. 5:387); GFP (green florescence protein), Chalfie, et al.,(1994) Science 263:802; luciferase, Teeri, et al., (1989) EMBO J. 8:343;and the maize genes encoding for anthocyanin production, Ludwig, et al.,(1990) Science 247:449.

The transformation vector comprising the particular regulatory sequencesof the present invention, operably linked to an isolated nucleotidesequence of interest in an expression cassette, can be used to transformany plant. In this manner, genetically modified plants, plant cells,plant tissue, seed, and the like can be obtained. Transformationprotocols can vary depending on the type of plant or plant cell, i.e.,monocot or dicot, targeted for transformation. Suitable methods oftransforming plant cells include microinjection, Crossway, et al.,(1986) Biotechniques 4:320-334; electroporation, Riggs, et al., (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606; Agrobacterium-mediatedtransformation, see for example, Townsend, et al., U.S. Pat. No.5,563,055; direct gene transfer, Paszkowski, et al., (1984) EMBO J.3:2717-2722; and ballistic particle acceleration, see for example,Sanford, et al., U.S. Pat. No. 4,945,050; Tomes, et al., (1995) in PlantCell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg andPhillips (Springer-Verlag, Berlin); and McCabe, et al., (1988)Biotechnology 6:923-926. Also see, Weissinger, et al., (1988) AnnualRev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926(soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein,et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein,et al., (1988) Biotechnology 6:559-563 (maize); Klein, et al., (1988)Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology8:833-839; Hooydaas-Van Slogteren, et al., (1984) Nature (London)311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman, et al. (Longman, NewYork), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports9:415-418; and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D. Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou, et al., (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells that have been transformed can be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants can then be grown andpollinated with the same transformed strain or different strains, andresulting plants having seed-preferred expression of the desiredphenotypic characteristic can then be identified. Two or moregenerations can be grown to ensure that seed-preferred expression of thedesired phenotypic characteristic is stably maintained and inherited.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention. Itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook, et al., (1989) MOLECULAR CLONING:A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated, size separation of fragments in the examplesbelow was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook, et al., (1989)MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. and numerous other referencessuch as, for instance, by Goeddel, et al., Nucleic Acids Res. 8:4057(1980).

Unless described otherwise, ligations were accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase (“ligase”) per 0.5 microgram of DNA.

Example 1 Construction of Vectors System for Temporal and Spatial SeedPreferred Expression of Cytokinin Biosynthetic Enzymes

Construction of PHP 11466 and PHP 11467 and Their Cointegrates (PHP11551and PHP11552. Respectively).

PHP 11466 and PHP 11467 were employed in particle gun transformationprotocols even though they have the right and left border for the tDNA.The versions designated PHP11551 and PHP11552 were used in Agro-mediatedtransformation protocols.

The ipt coding sequence was obtained as a 732 bp BamHI/HpaI fragment andinserted into a GLB1 expression cassette (BamHI/HpaI, 4.9 kb) to givePHP11310. The maize GLB1 promoter (Genbank Accession #L22344 L22295) andterminator (Genbank Accession #L22345 L22295) in PHP3303 comprise theGLB1 expression cassette. The pGLB1:ipt:GLB1 3′ cassette was moved astwo pieces (HindIII/BamHI 1401 bp and BamHI/EcoRI 1618 bp) into a T-DNAvector digested with EcoRI+HindIII (6.33 kb) to give PHP11363. Finally,a selectable marker gene (pUBI:UBIINTRON1:maize-optimized PAT:35S 3′)was added as a 2.84 kb HindIII fragment into HindIII-digested PHP11363(9.35 kb). In PHP11466, the two genes are in opposite orientationrelative to each other. In PHP11467, the two genes are oriented in thesame direction. After triparental mating the cointegrate ofPHP11466/PHP10523 was designated PHP11551. Likewise, the cointegrate ofPHP11467/PHP10523 was designated PHP11552.

Construction of PHP11404 and PHP11550

PHP 11404 was used with the biolistics-mediated transformation protocol.The plasmid has all the features of the Agro version. The plasmid thatwas actually used with the Agro-mediated transformation protocols was isPHP11550.

Using the plasmid PHP9063 (pUBI:UBIINTRON1:ipt:pinII 3′), an NcoIrestriction site was created at the start codon of ipt usingsite-directed mutagenesis (specifically, the MORPH™ Kit of 5 Prime→3Prime, Inc.). The resulting plasmid was designated PHP11362. The iptcoding sequence was then moved as a 724 bp NcoI/HpaI fragment intoPHP8001 (BamHI-cut, treated with Klenow to fill in the overhang to ablunt then cut with NcoI, 4.9 kb) to give PHP11401. PHP8001 contains theGZ-W64A promoter and terminator from the 27 KD zein gene of Z. mays(Genbank Accession #S78780). PHP11401 was digested with PacI+KpnI and a1.35 kb fragment inserted into PHP11287 (PacI/KpnI-digested, 10.87 kb)to give PHP11404. PHP11287 is a T-DNA vector that already carries theabove-described pUBI:UBIINTRON1:maize-optimized PAT:35S 3′ selectablemarker. After triparental mating the cointegrate of PHP11404/PHP10523was designated PHP11550.

Construction of PHP12975

The CIM1 promoter is described in U.S. patent application Ser. No.09/377,648, filed Aug. 19, 1999. Site-directed mutagenesis was used tocreate an NcoI site at the CIM1 translational start (PHP12699). Thepromoter was cut out as a 1.69 kb SacI/NcoI fragment and ligated to theipt coding sequence and pinII terminator from PHP11362 to form PHP12800.The CIM1:ipt:pinII transcriptional unit was then moved as a 2.8 kbBstEII fragment into BstEII-digested PHP12515 (9.5 kb), a binary vectoralready carrying the UBI:UBIINTRON1:MO-PAT:35S selectable marker betweenthe border sequences. The resulting plasmid was designated PHP12866.Triparental mating into A. tumefaciens LBA4404 (PHP10523) gave thecointegrate plasmid PHP12975.

Construction of PHP12425

Plasmid PHP11404 (described above) was used as a starting plasmid toreplace the GZ-W64A promoter with the LTP2 promoter from H. vulgare.PHP11404 DNA was digested with NotI and KpnI (9.46 kb fragment) andseparately with NcoI plus KpnI (1.24 kb fragment). These two fragmentswere mixed with a 1.52 kb NotI/NcoI fragment from PHP8219 containing theLTP2 promoter and ligated. The resulting plasmid product was designatedPHP12333. Triparental mating of this plasmid into A. tumefaciens LBA4404(PHP10523) gave the cointegrate plasmid PHP12425.

Triparental Mating and Selectable Marker 35s:bar:pinII:

All vectors were constructed using standard molecular biologytechniques. The T-DNA region for transformation consists of the T-DNAborder sequences flanking a reporter gene and a selectable marker. Thereporter is inserted proximal to the right T-DNA border and consists ofthe 2.0 kb PstI fragment of the maize ubiquitin promoter Ubi-1(Christensen, et al., 1992) with flanking 5′ HindIII and 3′ BamHIrestriction sites. The ubiquitin promoter was ligated to the 5′ BamHIsite of a beta-glucuronidase (GUS) reporter gene (Jefferson, et al.,1986), containing the second intron from potato ST-LS1 (Vancanneyt, etal., 1990). The potato proteinase II (pinII) terminator (bases 2 to 310from An, et al., (1989) Plant Cell 1(1):115-122) was blunt-end ligateddownstream of the GUS coding sequence. On the 3′ end of the terminatoris a NotI restriction site.

The selectable marker consists of an enhanced cauliflower mosaic virus35S promoter (bases −421 to −90 and −421 to +2 from Gardner, R. C., etal., (1981) Nucl. Acids Res. 9:2871-88) with a flanking 5′ NotI site and3′ PstI site. A PstI/SalI fragment containing the 79 bp tobacco mosaicvirus leader (Gallie, D. R., et al., (1987) Nucl. Acids Res. 15:3257-73)is inserted downstream of the promoter followed by a SalI/BamHI fragmentcontaining the first intron of maize alcohol dehydrogenase ADH1-S(Dennis, et al., 1984). The BAR coding sequence (Thompson, C. J., etal., (1987) Embo J. 6:2519-23) was cloned into the BamHI site, with thepinII terminator ligated downstream. The pinII signal is flanked by a 3′SacI site.

The T-DNA of PHP8904 was integrated into the super binary plasmid pSB1(Ishida, et al., 1996) by homologous recombination between the twoplasmids. E. coli strain HB101 containing PHP8904 was mated withAgrobacterium strain LBA4404 harboring pSB1 to create the cointegrateplasmid in Agrobacterium designated LBA4404 (PHP10525) (by the method,Ditta, G., et al., (1980) Proc. Natl. Acad. Sci. USA 77:7347-51) LBA4404(PHP10525) was selected for by Agrobacterium resistance to spectinomycinand verified as a recombinant by a SalI restriction digest of theplasmid.

Example 2 Transformation of Maize

Biolistics:

The inventive polynucleotides contained within a vector are transformedinto embryogenic maize callus by particle bombardment, generally asdescribed by Tomes, D. et al., (1995) Plant Cell, Tissue and OrganCulture: Fundamental Methods, Eds. O. L. Gamborg and G. C. Phillips,Chapter 8, pgs. 197-213 and is briefly outlined below. Transgenic maizeplants are produced by bombardment of embryogenically responsiveimmature embryos with tungsten particles associated with DNA plasmids.The plasmids consist of a selectable and an unselected structural gene.

Preparation of Particles:

Fifteen mg of tungsten particles (General Electric), 0.5 to 1.8μ,preferably 1 to 1.8μ, and most preferably 1μ, are added to 2 ml ofconcentrated nitric acid. This suspension was sonicated at 0° C. for 20minutes (Branson Sonifier Model 450, 40% output, constant duty cycle).Tungsten particles are pelleted by centrifugation at 10000 rpm (Biofuge)for one minute, and the supernatant is removed. Two milliliters ofsterile distilled water are added to the pellet, and brief sonication isused to resuspend the particles. The suspension is pelleted, onemilliliter of absolute ethanol is added to the pellet, and briefsonication is used to resuspend the particles. Rinsing, pelleting, andresuspending of the particles is performed two more times with steriledistilled water, and finally the particles are resuspended in twomilliliters of sterile distilled water. The particles are subdividedinto 250-ml aliquots and stored frozen.

Preparation of Particle-Plasmid DNA Association:

The stock of tungsten particles are sonicated briefly in a water bathsonicator (Branson Sonifier Model 450, 20% output, constant duty cycle)and 50 ml is transferred to a microfuge tube. All the vectors were cis:that is the selectable marker and the gene of interest were on the sameplasmid. These vectors were then transformed either singly or incombination.

Plasmid DNA was added to the particles for a final DNA amount of 0.1 to10 μg in 10 μL total volume, and briefly sonicated. Preferably, 10 μg (1μg/μL in TE buffer) total DNA is used to mix DNA and particles forbombardment. Specifically, 1.0 μg of PHP 11404, 11466, and/or 11467 (1μg/μL), where any cytokinin biosynthetic enzyme polynucleotide canreplace ipt were used per bombardment. Fifty microliters (50 μL) ofsterile aqueous 2.5 M CaCl₂ are added, and the mixture is brieflysonicated and vortexed. Twenty microliters (20 μL) of sterile aqueous0.1 M spermidine are added and the mixture is briefly sonicated andvortexed. The mixture is incubated at room temperature for 20 minuteswith intermittent brief sonication. The particle suspension iscentrifuged, and the supernatant is removed. Two hundred fiftymicroliters (250 μL) of absolute ethanol are added to the pellet,followed by brief sonication. The suspension is pelleted, thesupernatant is removed, and 60 ml of absolute ethanol are added. Thesuspension is sonicated briefly before loading the particle-DNAagglomeration onto macrocarriers.

Preparation of Tissue

Immature embryos of maize variety High Type II are the target forparticle bombardment-mediated transformation. This genotype is the F₁ oftwo purebred genetic lines, parents A and B, derived from the cross oftwo know maize inbreds, A188 and B73. Both parents are selected for highcompetence of somatic embryogenesis, according to Armstrong, et al.,(1991) Maize Genetics Coop. News 65:92 (1991).

Ears from F₁ plants are selfed or sibbed, and embryos are asepticallydissected from developing caryopses when the scutellum first becomesopaque. This stage occurs about 9-13 days post-pollination, and mostgenerally about 10 days post-pollination, depending on growthconditions. The embryos are about 0.75 to 1.5 millimeters long. Ears aresurface sterilized with 20-50% Clorox for 30 minutes, followed by threerinses with sterile distilled water.

Immature embryos are cultured with the scutellum oriented upward, onembryogenic induction medium comprised of N6 basal salts, Erikssonvitamins, 0.5 mg/l thiamine HCl, 30 gm/l sucrose, 2.88 gm/l L-proline, 1mg/l 2,4-dichlorophenoxyacetic acid, 2 gm/l Gelrite, and 8.5 mg/l AgNO₃.Chu, et al., (1975) Sci. Sin. 18:659; Eriksson, (1965) Physiol. Plant18:976. The medium is sterilized by autoclaving at 121° C. for 15minutes and dispensed into 100×25 mm Petri dishes. AgNO₃ isfilter-sterilized and added to the medium after autoclaving. The tissuesare cultured in complete darkness at 28° C. After about 3 to 7 days,most usually about 4 days, the scutellum of the embryo swells to aboutdouble its original size and the protuberances at the coleorhizalsurface of the scutellum indicate the inception of embryogenic tissue.Up to 100% of the embryos display this response, but most commonly, theembryogenic response frequency is about 80%.

When the embryogenic response is observed, the embryos are transferredto a medium comprised of induction medium modified to contain 120 gm/lsucrose. The embryos are oriented with the coleorhizal pole, theembryogenically responsive tissue, upwards from the culture medium. Tenembryos per Petri dish are located in the center of a Petri dish in anarea about 2 cm in diameter. The embryos are maintained on this mediumfor 3-16 hour, preferably 4 hours, in complete darkness at 28° C. justprior to bombardment with particles associated with plasmid DNAscontaining the selectable and unselectable marker genes.

To effect particle bombardment of embryos, the particle-DNA agglomeratesare accelerated using a DuPont PDS-1000 particle acceleration device.The particle-DNA agglomeration is briefly sonicated and 10 ml aredeposited on macrocarriers and the ethanol is allowed to evaporate. Themacrocarrier is accelerated onto a stainless-steel stopping screen bythe rupture of a polymer diaphragm (rupture disk). Rupture is effectedby pressurized helium. The velocity of particle-DNA acceleration isdetermined based on the rupture disk breaking pressure. Rupture diskpressures of 200 to 1800 psi are used, with 650 to 1100 psi beingpreferred, and about 900 psi being most highly preferred. Multiple disksare used to effect a range of rupture pressures.

The shelf containing the plate with embryos is placed 5.1 cm below thebottom of the macrocarrier platform (shelf #3). To effect particlebombardment of cultured immature embryos, a rupture disk and amacrocarrier with dried particle-DNA agglomerates are installed in thedevice. The He pressure delivered to the device is adjusted to 200 psiabove the rupture disk breaking pressure. A Petri dish with the targetembryos is placed into the vacuum chamber and located in the projectedpath of accelerated particles. A vacuum is created in the chamber,preferably about 28 in Hg. After operation of the device, the vacuum isreleased and the Petri dish is removed.

Bombarded embryos remain on the osmotically-adjusted medium duringbombardment, and 1 to 4 days subsequently. The embryos are transferredto selection medium comprised of N6 basal salts, Eriksson vitamins, 0.5mg/1 thiamine HCl, 30 gm/l sucrose, 1 mg/l 2,4-dichlorophenoxyaceticacid, 2 gm/l Gelrite, 0.85 mg/l Ag NO₃ and 3 mg/l bialaphos (Herbiace,Meiji). Bialaphos is added filter-sterilized. The embryos aresubcultured to fresh selection medium at 10 to 14 day intervals. Afterabout 7 weeks, embryogenic tissue, putatively transformed for bothselectable and unselected marker genes, proliferates from about 7% ofthe bombarded embryos. Putative transgenic tissue is rescued, and thattissue derived from individual embryos is considered to be an event andis propagated independently on selection medium. Two cycles of clonalpropagation are achieved by visual selection for the smallest contiguousfragments of organized embryogenic tissue.

A sample of tissue from each event is processed to recover DNA. The DNAis restricted with a restriction endonuclease and probed with primersequences designed to amplify DNA sequences overlapping the cytokininbiosynthetic enzymes and non-cytokinin biosynthetic enzyme portion ofthe plasmid. Embryogenic tissue with amplifiable sequence is advanced toplant regeneration.

For regeneration of transgenic plants, embryogenic tissue is subculturedto a medium comprising MS salts and vitamins (Murashige & Skoog, (1962)Physiol. Plant 15:473), 100 mg/l myo-inositol, 60 gm/l sucrose, 3 gm/lGelrite, 0.5 mg/l zeatin, 1 mg/l indole-3-acetic acid, 26.4 ng/lcis-trans-abscissic acid, and 3 mg/l bialaphos in 100×25 mm Petridishes, and is incubated in darkness at 28° C. until the development ofwell-formed, matured somatic embryos can be seen. This requires about 14days. Well-formed somatic embryos are opaque and cream-colored, and arecomprised of an identifiable scutellum and coleoptile. The embryos areindividually subcultured to a germination medium comprising MS salts andvitamins, 100 mg/l myo-inositol, 40 gm/l sucrose and 1.5 gm/l Gelrite in100×25 mm Petri dishes and incubated under a 16 hour light:8 hour darkphotoperiod and 40 meinsteinsm⁻²sec⁻¹ from cool-white fluorescent tubes.After about 7 days, the somatic embryos have germinated and produced awell-defined shoot and root. The individual plants are subcultured togermination medium in 125×25 mm glass tubes to allow further plantdevelopment. The plants are maintained under a 16 hour light:8 hour darkphotoperiod and 40 meinsteinsm⁻²sec⁻¹ from cool-white fluorescent tubes.After about 7 days, the plants are well-established and are transplantedto horticultural soil, hardened off, and potted into commercialgreenhouse soil mixture and grown to sexual maturity in a greenhouse. Anelite inbred line is used as a male to pollinate regenerated transgenicplants.

Agrobacterium-Mediated:

When Agrobacterium-mediated transformation is used, the method of Zhaois employed as in PCT patent publication WO98/32326, the contents ofwhich are hereby incorporated by reference. Briefly, immature embryosare isolated from maize and the embryos contacted with a suspension ofAgrobacterium (step 1: the infection step). In this step the immatureembryos are preferably immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Preferably theimmature embryos are cultured on solid medium following the infectionstep. Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step) and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

Example 3 Identification of High Cytokinin Transgenic Corn Lines

The resulting transformants are screened for elevated levels ofcytokinin using a combination of direct measurements and in vivocorrelates.

Vivipary Experiments (glb1:ipt Constructs):

Because it is appreciated that seed dormancy is controlled by the ratioof ABA:cytokinin, an elevated cytokinin level in the seed could induce aviviparous phenotype.

Glb1::ipt transformants were initiated using GS3 embryos and eitherAgrobacterium-(inventive polynucleotides 11551 and 11552) orbiolistic-(inventive polynucleotides 11466 and 11467) mediatedtransformation. Plantlets were regenerated 2-3 months later and theseplantlets (T0's) were transferred to the greenhouse after an additional2-3 months. At anthesis, T0's were crossed with HG11 and vivipary wasdetected in the developing T1 seed approximately 30 days later.Developing T1 seed that exhibited the viviparous phenotype was rescuedby replanting without seed drying. Viable plants were analyzed by PCRand leaf-painting to determine if the ipt gene and the selectable marker(PAT gene) were present. T1 plants flowered and ears were selfed tocreate T2 seed. Those plants carrying the ipt gene (PCR- and leafpaint-positive) produced seed that were segregating 3:1 for the gene,whereas the plants that were PCR- and leaf paint-negative did notsegregate.

Cytokinin Determinations:

At 19 and 23 days after pollination (DAP), ten seeds were harvested fromeach of four replications per event (11551 and 11552). Seeds were thenseparated into embryo and endosperm and frozen in liquid nitrogen. Ateach sampling date, embryo tissue from the four replications was pooledand cytokinin levels were determined. Endosperm tissue was processed inan similar manner. The results are presented in FIG. 1.

glb1::ipt Seed Propagation:

In order to propagate the viviparous seed, half of the remaining plantswithin each event were harvested at 25 DAP. Ears were placed in dryerboxes and ambient air (22 to 25° C.) was blown across them for threedays to slowly dry the seed. Dryer boxes containing the transgenic earswere then transferred to a growth chamber and seeds were dried to ˜12%moisture by blowing 35° C. air across them for 3 to 5 days. Individualears were then shelled and the seeds stored at 10° C. and 50% RH.

Phenotype Determination:

To determine the proportion of seed exhibiting vivipary, ears from theremaining half of the plants were harvested at approximately 45 DAP andseed scored for degree of vivipary. The four classes of vivipary weredefined as:

Class 1: No apparent swelling of coleoptile.

Class 2: Visible swelling of coleoptile, but no elongation.

Class 3: Visible swelling of coleoptile with elongation past thescutellum, but no rupture of pericarp.

Class 4: Visible swelling of coleoptile with elongation past thescutellum and rupture of pericarp.

The results are shown below in Table 1.

TABLE 1 Vivipary Characterization at 45 T2 Phenotype DAP Event SID # PCRResult Leaf Paint Result Vivipary Result Class 1 Class 2 Class 3 Class 4Total Seed # 11551 751412 + + + 4 4 7 4 19 751415 + + + 0 169 34 2 205751416 + + + 99 28 18 58 204 751417 + + + 167 55 39 18 279 751420 + + +2 193 47 0 242 751422 + + + 0 141 93 11 245 Sum 272 590 238 94 119411551 751425 + + + 50 57 85 4 196 751426 + + + 0 75 64 12 151761429 + + + 66 40 19 4 129 751432 + + + 41 16 14 4 75 Sum 157 188 18224 991 11551 751433 − − 751434 − − − 438 0 0 0 439 751435 − − − 405 0 00 405 751436 − − − 375 0 0 0 375 Sum 405 0 0 0 405 11551 751437 + + + 1450 78 19 161 751438 − + + 52 37 128 10 227 751439 + + + 128 92 101 9 330751443 + + + 70 84 89 4 247 Sum 264 263 396 42 965 11551 751441 − − −375 0 0 0 375 751442 + − 751444 − − − 343 0 0 0 343 Sum 359 0 0 0 35911551 751445 + 158 76 38 9 281 751448 − + + 4 126 79 3 212 751450 + + +101 83 44 14 242 751451 + + + 101 33 48 1 183 Sum 364 318 209 27 91011552 752902 + + 16 53 62 4 135 752908 + + + 35 74 24 4 137 752810 + + +9 132 14 0 155 752911 + + + 2 148 39 3 192 752912 + + + 0 40 27 2 69752913 + + + 49 36 36 8 129 752814 + + + 75 47 12 21 155 762919 + + 2572 80 16 193 Sum 211 602 294 58 1165 11551 752924 + + + 109 57 98 16 280752930 + + + 6 27 22 10 65 752936 + + + 53 60 47 3 163 762937 + + + 5336 70 10 169 752939 + + + 58 48 68 6 180 752940 + + 0 1 0 0 Sum 279 229305 45 857The results of the phenotypic evaluation demonstrated that the presenceof the ipt gene resulted in a greater occurrence of vivipary (Classes 2through 4), relative to the plants without the gene.Increased Seed Dry Unit Mass (gz:ipt Constructs):

Because kernel mass is a function of the number of endosperm cells andamyloplasts, and cytokinins have been implicated in increasing endospermcell number and in the differentiation of amyloplasts from proplastids,seeds exhibiting an increased level of cytokinin should yield acorresponding increase in seed dry unit mass.

Gz::ipt transformants were initiated using GS3 embryos andAgrobacterium-mediated transformation (inventive polynucleotide 11550).Plantlets were regenerated in 2-3 months in 1997 and these plantlets(T0's) were transferred to the greenhouse after an additional 2-3months. At anthesis, T0's were crossed with HG11 and at maturity theears were harvested, shelled and the seed used for additional seedpropagation (both backcrossing to HG11 and self-pollinating). T2 seed(both BC2 generation and selfs) was then planted. The T2 plants wereanalyzed by using PCR and leaf painting to determine if the ipt gene andthe selectable marker (PAT gene) were present, respectively. Subsets ofthese plants were self-pollinated for cytokinin determinations, orallowed to open pollinate for phenotype determinations (yield and yieldcomponents).

Cytokinin Determinations:

Samples can be collected and analyzed as follows. At 10, 16 and 22 DAP,50 to 100 seeds can be collected from two replications per event (eachreplication was composed of two subsamples) and the pedicel removed. Forthe 10 DAP samples, the remaining seed tissue can be placed directlyinto liquid nitrogen (tissue defined as “seed,” composed primarily ofpericarp, aleurone, endosperm and nucellus). In contrast, at 16 and 22DAP, the embryo can be first dissected from the remaining seed tissue(tissue defined as “seed minus embryo,” and composed primarily ofpericarp, aleurone and endosperm) and then both tissues placed directlyinto liquid nitrogen.

Phenotype Determination:

To determine the effect of the gz::ipt construct on seed mass,individual plants are hand harvested at physiological maturity (visibleblack layer), the seed shelled and oven dried to a constant mass (104°C., minimum of 3 days). Yield (g plant) and the components of yield(ears per plant, seeds per ear and wt per seed) are determined onprimary and secondary ears.

Increased Frequency of Seed Set and Increased Number of Seeds

(Itp2:ipt Constructs):

Because yield is a combination of both frequency of seed set and numberof seeds per ear, seeds exhibiting an increased level of cytokinin inthe early stages of seed set and formation should have ears with acorresponding increase in seed set and numbers.

Ltp2::ipt transformants were initiated using GS3 embryos andAgrobacterium-mediated transformation (12425). Plantlets wereregenerated in 2-3 months in 1998 and these plantlets (T0's) weretransferred to the greenhouse after an additional 2-3 months. Atanthesis, T0's were crossed with HG11 and at maturity the ears wereharvested, shelled and the seed used for additional seed propagation(both backcrossing to HG11 and self-pollinating). The number of seedsper T0 event, and the number of events which set seed were compared to anumber of other transgenic events with promoter:gene combinations otherthan Itp2:ipt. These are shown in Table 2.

TABLE 2 Seed set average of T0 events of ltp2:ipt gene compared to othergenes in T0 plants grown under identical green house conditions in 1998in Johnston, IA. Inventive gene number average # polynucleotidedescription T0′s % T0 w/seed seeds 12425 ltp2:ipt 35 82.9 198 12384lignin 92 22.8 145 12417 carbohydrate 40 55.0 156 12427 maturity 35 45.769 12428 lignin 29 75.9 174 12723 lignin 35 62.9 184 12724 lignin 3545.7 161

Compared to % seed set and average # seeds per T0 plant, Itp2:ipt, hadboth the highest % of T0 plants which set seed and the highest numericalaverage # of seeds compared to six other transgenic combinations in T0plants grown at the same time and under the same greenhouse conditions.These results indicate that expression of cytokinin in the aleuronelayer of early seed development may increase yield by increasing boththe percentage of plants that set seed, and the number of seeds set perear.

Subsequent generations will be grown at different field locations todetermine their seed set and seed number characteristics and seed yieldcompared to non-transgenic controls of the same genetic background.Cytokinin levels will also be measured on transgenic and non-transgenickernels of similar genetic background.

Cytokinin Determinations:

Samples can be collected and analyzed as follows. At 2, 6 and 22 DAP, 50to 100 seeds can be collected from two replications per event (eachreplication composed of two subsamples) and the pedicel removed. For the2, 6, and 22 DAP samples, the remaining seed tissue can be placeddirectly into liquid nitrogen (tissue defined as “seed,” composedprimarily of pericarp, aleurone, endosperm and nucellus).

Example No. 4 Isolation of ipt and Isolation of ckx1-2

Briefly, PCR primers preferably containing convenient restrictionendonuclease sites are constructed. Two useful primers are shown below:

SEQ ID NO: 38 (Upper primer with Bam HI site)5′caucaucaucauggatccaccaatggatctacgtctaattttcggtcc aac 3′ SEQ ID NO: 39(Lower primer with HpaI site)5′cuacuacuacuagttaactcacattcgaaatggtggtccttc 3′

The introduced restriction sites are bolded. The portion of the primerthat binds to the template extends from nucleotides 22 and 19 to the 3′terminus, respectively. A BamHI site “ggatcc” (bolded) and a Kozakconsensus sequence were introduced before the start codon and a HpaIsite “gttaac” (also bolded) was introduced after the stop.

The Agrobacterium tumefaciens strain carrying the tumor-inducing plasmidpTi Bo542 was obtained (See, Guyon, P., et al., (1980) Agropine innull-type crown gall tumors: Evidence for generality of the opineconcept, Proceedings of the National Academy of Sciences (U.S.)77(5):2693-97; Chilton, W. S., et al., (1985) Absolute stereochemistryof leucinopine, a crown gall opine, Phytochemistry (Oxford)24(2):221-24; Strabala, T. J., et al., (1989) Isolation andcharacterization of an ipt gene from the Ti plasmid Bo542, Molecular &General Genetics 216:388-94) and live bacteria were used for the PCRtemplate. Standard PCR conditions were used. An example of suchconditions follows: Volume per reaction of 100 μL, with 0.5 μL of 10ng/μL target plasmid, 0.05 Unit/μL Taq Polymerase, 0.5 μM each ofprimers, 0.8 mM dNTP's 1× Buffer in a thin walled tube. Mix reagents,keep on ice. Add target plasmid to tube and then add the 100 μL ofreaction mix to each tube. Pre-incubate in a thermocycler at 95° C. for3 minutes. Then cycle five times at 95° C. for 35 seconds, 55° C. for 1minute, and 72° C. for 1 minute. Follow with 30 cycles at 95° C. for 35seconds, 65° C. for 1 minute, and 72° C. for 1 minute. Finalize reactionby dwelling for 10 minutes at 72° C. and allowing to soak at 6° C. PCRproduct was then cloned into DH5α cells using a kit made by LifeTechnologies according to manufacturer's instructions. DNA was extractedfrom putative transformants, cut with BamHI and HpaI, and run on gel toconfirm transformation. This insert was then gel purified andtransformed into a convenient expression vector, such as 7921 vector DNAcontaining a Ubi promoter and pinII terminator.

A preferred DNA sequence is provided in Molecular and General Genetics216:388-394 (1989). It contains an open reading frame encoding a proteinof 239 amino acid residues, with a deduced molecular weight of about26.3 kDa (Calculated as the number of amino acid residues×110).

Isolation of Maize Cytokinin Oxidase Gene, cytox 1-2

Another preferred DNA sequence is set out below as SEQ. I.D. NO: 1. Itcontains an open reading frame encoding a protein of about 535 aminoacid residues, SEQ ID NO.: 2, with a deduced molecular weight of about58.9 kDa (Calculated as the number of amino acid residues×110). A copyof cytokinin oxidase can be prepared synthetically employing DNAsynthesis protocols well known to those skilled in the art of genesynthesis. Alternatively, a copy of the gene may be isolated directlyfrom a cytokinin oxidase harboring organism by PCR cloning. A maizecytokinin oxidase gene (ckx1) was cloned by Roy Morris of the Universityof Missouri and the sequence deposited in Genbank. (Morris, et al.,(1999) Isolation of a gene encoding a glycosylated cytokinin oxidasefrom maize. Biochem. Biophys. Res. Commun. 255(2):328-333. See also,Houba-Herin, et al., (1999) Cytokinin oxidase from Zea mays:purification, cDNA cloning and expression in moss protoplasts. Plant J.(6):615-626). PCR primers preferably containing convenient restrictionendonuclease sites are constructed. Two useful primers are shown below:

(SEQ ID NO: 42) 5′ CATGCCATGGCGGTGGTTTATTACCTGCT 3′ (with NocI site at5′ end) (SEQ ID NO: 43) 5′ CGGGATCCTCATCATCAGTTGAAGATGTCCT 3′ (withBamHI site at 3′ end)

These primers were designed against the sequence of ckx1 and reversetranscriptase PCR (RT-PCR) was utilized to isolate cytokinin oxidasegenes from several different tissues of developing maize kernels. DNAfragments were amplified from the following tissues: 10 DAP, 13 DAP, 18DAP, and 20 DAP endosperms; as well as 10 DAP, 18 DAP, and 20 DAPembryos, where DAP is days after pollination. Fragments from all tissuesmigrated to 1.6 Kb in the gel, which is equal to that of the publishedsequence. We selected one of the fragments (from 18 DAP embryos) andsequenced the DNA. This fragment is referred to herein as Cytox1-2 andits full-length sequence is set out below in SEQ ID NO.: 1. At the aminoacid level, there is a 98% homology between the ckx1 gene and cytox1-2,therefore, one of skill in the art would recognize that cytox1-2 is acytokinin oxidase gene from maize.

Example 5 Expression of Transgenes in Monocots

A plasmid vector is constructed comprising the Zag2.1 promoter (SEQ IDNO: 3) or Zap promoter (SEQ ID NO: 5, also known as ZmMADS) or tb1promoter (SEQ ID NO: 17) operably linked to a an isolated polynucleotideencoding ipt (SEQ ID NO: 1). This construct can then be introduced intomaize cells by the following procedure.

Immature maize embryos are dissected from developing caryopses derivedfrom crosses of maize lines. The embryos are isolated 10 to 11 daysafter pollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu, et al., (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus, consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structures,proliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid p35S/Ac (Hoechst Ag, Frankfurt, Germany) or equivalent maybe used in transformation experiments in order to provide for aselectable marker. This plasmid contains the Pat gene (see, EuropeanPatent Publication 0 242 236) which encodes phosphinothricin acetyltransferase (PAT). The enzyme PAT confers resistance to herbicidalglutamine synthetase inhibitors such as phosphinothricin. The pat genein p35S/Ac is under the control of the 35S promoter from CauliflowerMosaic Virus (Odell, et al., (1985) Nature 313:810-812) and comprisesthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein, et al., (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovers a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm, et al., (1990) Bio/Technology 8:833-839).

Example 6 Expression of Transgenes in Dicots

Soybean embryos are bombarded with a plasmid comprising the Zag2.1promoter operably linked to a heterologous nucleotide sequence encodingipt, as follows. To induce somatic embryos, cotyledons of 3-5 mm inlength are dissected from surface-sterilized, immature seeds of thesoybean cultivar A2872, then cultured in the light or dark at 26° C. onan appropriate agar medium for six to ten weeks. Somatic embryosproducing secondary embryos are then excised and placed into a suitableliquid medium. After repeated selection for clusters of somatic embryosthat multiply as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with fluorescentlights on a 16:8 hour day/night schedule. Cultures are sub-culturedevery two weeks by inoculating approximately 35 mg of tissue into 35 mlof liquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette of interest, comprising the Zag2.1promoter and a heterologous polynucleotide encoding ipt, can be isolatedas a restriction fragment. This fragment can then be inserted into aunique restriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Analysis of Ear Growth Rate of T1 (D2F1 Hemizygous) SlantsUnder Non-Stress Conditions

Transformation of maize with the zag2.1::ipt construct was performed asdescribed in Example 5. Regenerated plants were pollinated with one ofthe parent genotypes to create D2F1 seed (D2 referring to two doses of aparent; also known as T1 seed). Of 17 original transformants, nine wereselected for advancement based on favorable genetic complexity (i.e.,single- to low-copy number as determined by Southern blot analysis),intactness of the plant transcriptional unit (as determined by Southernblot analysis), and adequate seed numbers.

The D2F1 seed was planted in a replicated, well-watered field trial inJohnston, Iowa. Dry mass of unpollinated ears was measured at initialsilk emergence and seven days later. Ear growth rate (EGR) wascalculated as the difference in dry mass divided by the number of days.As shown in FIG. 2, four of the nine events tested showed an increase inear growth rate, relative to transgene-negative sibs planted ascontrols. Presence of the ipt transcript in developing ears representingall nine events was confirmed via RT-PCR.

However, space constraints in the field prohibited direct comparisons oftransgene-positive and transgene-negative plants of the same event andsame genotype. Instead, control plants in this example were grown from abulked sample of segregating T1 seed. Control plots were thinned tostandard density; also, transgene-positive plants, identified via leafpainting with herbicide, were rogued. As a result of the bulking acrossevents and genotypes, and the variation in field conditions fortransgenic vs. control plants, the differences in EGR between transgenicand control plants were muted and the results were inconclusive.Therefore, all nine events were carried forward for yield analysis thefollowing year.

Ear growth rate differences are expected for the transgenic events andcan be properly evaluated with direct comparisons in which geneticbackground and field growing conditions are held constant.

Example 8 Analysis of Yield of T2 (D3F1 Hemizygous) Plants UnderNon-Stress Conditions

T2 seed representing the nine selected events (derived from pollinationof T1 plants with a recurrent parent) was planted in an unreplicated,well-watered field trial in Johnston, Iowa. Presence of the ipttranscript in developing ears was confirmed via Northern blot analysis.Transgene-negative sibs of each event were planted as controls and apair-wise analysis of each event was conducted (a difference analysis)as well as an event average analysis. All subject plants were detasseledand pollinated by a mixed non-transgenic male parent.

Yield was determined by collecting primary ears. Grain was bulked byevent and by the presence or absence of the transgene; grain was thenoven-dried and measured for total dry mass. Results are shown in FIG. 3.Grain yield of seven of the nine events was greater than that ofcontrols. Kernel number, ear length, and kernel mass were also measured;results for transgenics exceeded those for non-transgenic sibs in fiveout of nine events for ear length; and in five out of nine events forboth kernel number and dry matter per kernel.

Example 9 Analysis of D4F3 Homozygous Plants for Yield and Plant Height

Next-generation progeny of the nine selected events were evaluated in areplicated, well-watered field trial in Johnston, Iowa, in 2002.Transgene-negative sibs were planted as controls. All subject plantswere detasseled; a mix of non-transgenic plants served as the pollensource.

Plant heights were measured at V10 and V12. (For growth stages, see, Howa Corn Plant Develops, Iowa State University of Science and TechnologyCooperative Extension Service Special Report No. 48, Reprinted June1993.) Five of the nine events showed a statistically significantincrease in plant height, as shown in FIG. 4.

Yield was determined by collecting all grain-bearing ears. Grain wasbulked as appropriate, oven-dried, and measured for total dry mass. Asshown in FIG. 5, three of the nine events showed a statisticallysignificant increase in yield, including two of the events also showingincreased plant height. Ear number, kernel number, and kernel mass werealso measured, as shown in FIG. 6.

Example 10 Analysis of Yield, Plant Height, Leaf Greenness, Biomass, andTransgene Expression of D4F3 Plants Under Drought Stress

Homozygous progeny of the nine events were evaluated under droughtconditions in a replicated field trial at Woodland, Calif., in 2002.Supplemental irrigation was withheld to target a stress during anthesissufficient to decrease yield 40% to 50%. To do this, water was withheldbeginning at 920 GDUs (growing degree units) post planting and resumedat 1860 GDUs. All subject plants were detasseled and pollinated by amixed non-transgenic male parent. Presence of the ipt transcript wasdetermined by Northern blot analysis of developing stem, leaf, andtassel.

Leaf greenness was measured approximately one week prior to floweringwith a Minolta SPAD chlorophyll meter. Plant height was measured at thesame time. Five of the nine events showed a statistically significantincrease in plant height, as shown in FIG. 7. Four of these five, andone additional event, showed increased leaf greenness, as shown in FIG.8.

Yield was determined by collecting all grain-bearing ears. Grain wasbulked as appropriate, oven-dried, and measured for total dry mass.Kernel number, ear number, and kernel mass were also measured. Three ofthe nine events gave improved yield results, as shown in FIG. 9; allthree of these events had also displayed increased plant height and leafgreenness. The increase in plant biomass for one of these events isshown in FIG. 10. In addition, in all events tested, the transgenepositive plants showed an increase in steady-state levels of ipttranscripts in various vegetative and reproductive tissues relative tothat in transgene negative plants.

Example 11 Analysis of Transgene Effect on Yield of in Non-StressConditions

Several constructs were tested for their impact upon yield in apreliminary screen at one location with supplemental irrigation asrequired. All constructs were evaluated as multiple events, dose 2 eliteparent, and tested for per se yield with two reps per event. Onlytransgene-positive plants were harvested and then all events werecompared against each other for their yield advantage. The results areshown in Table 2, where the different constructs are ranked by yield,highest at the top and lowest yielding at the bottom. The second columnrecords the raw yield, whereas the third column records the differencebetween that entry and the mean of all of the constructs.

TABLE 2 Construct minus mean PHP Bu/acr of other constructs bu/acr S.E.P value PHP19698 138.0 13.05 5.81 0.0248 PHP19020 137.4 12.54 5.540.0236 PHP19874 135.8 10.87 5.06 0.0318 PHP15418 132.0 9.00 1.85 <.0001PHP16036 131.2 6.23 4.86 0.2006 PHP19304 129.4 4.42 4.17 0.2895 PHP19369128.6 3.57 4.17 0.3925 PHP19512 126.4 1.30 4.17 0.7543 PHP19513 126.00.85 4.22 0.8399 PHP19815 124.3 −0.89 4.35 0.8375 PHP19380 124.1 −1.074.17 0.7977 PHP16889 124.0 −1.18 7.79 0.8794 PHP17897 123.3 −1.87 5.670.7416 PHP16037 122.9 −2.34 4.97 0.6378 PHP19699 122.7 −2.58 4.32 0.5497PHP18070 122.1 −3.11 4.97 0.5315 PHP16176 121.7 −3.52 5.22 0.5011PHP16178 121.5 −3.74 6.08 0.5385 PHP16172 120.8 −4.45 5.58 0.4258PHP19523 120.5 −4.82 4.20 0.2478 PHP19814 120.3 −5.04 4.37 .0.2504PHP19368 118.4 −6.92 4.17 0.0968 PHP19514 118.1 −7.31 4.20 0.0812PHP19822 117.4 −8.23 4.91 0.0936 PHP18016 114.5 −10.02 4.99 0.0288PHP19370 114.0 −11.56 4.20 0.0058 PHP19303 108.9 −10.00 4.17 <.0001

It can be seen that four constructs at the top of the table weresignificantly higher yielding than any of the other constructs tested.Similarly, three constructs exhibited a significantly lower yield thanany of the other constructs in this test. The remainder was notsufficiently distinguished from each other and it can be assumed thattheir transgene does not create an impact obviously different from justthe background genotype. In this test, there were four IPT constructsthat were driven in expression by either Zag2.1 or Zap and this grouprepresent the four highest yielding constructs in this test: PHP19698Zap::IPT, PHP19020 Zag:IPT with Ubi:BAR, PHP19874 Zag::IPT with 35s BAR(head-to-head), and PHP15418 the original Zag::IPT construct with 35sBAR (head-to-tail). While the rest of these constructs were tested with10 events per construct, in fact the re-make of PHP15418 contained over90 events. These results clearly show the impact of coupling the IPTgene with a promoter/regulatory sequence with expression focused aroundfemale meristems.

Example 12 Analysis of zag2:ipt Expression in Soybean

Soybean embryogenic suspension cultures were transformed by the methodof particle gun bombardment using procedures known in the art (Klein, etal., (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050; Hazel,et al., (1998) Plant Cell. Rep. 17:765-772; Samoylov, et al., (1998) InVitro Cell Dev. Biol.-Plant 34:8-13).

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA, or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. In this example, the recombinantDNA fragments were isolated from the entire plasmid before being usedfor bombardment. For every eight bombardments of soybean tissue, 30 μlof solution were prepared with 3 mg of 0.6 μm gold particles and up to100 picograms (pg) of DNA fragment per base pair of DNA fragment.

The soybean transformation experiments were carried out using tworecombinant DNA fragments. The recombinant DNA fragment used to expressthe IPT gene was on a separate recombinant DNA fragment from theselectable marker gene providing resistance to sulfonylurea herbicides.Both recombinant DNA fragments were co-precipitated onto gold particles.

Stock tissue for these transformation experiments was obtained byinitiation from soybean immature seeds. Secondary embryos were excisedfrom explants after 6 to 8 weeks on culture initiation medium. Theinitiation medium was an agar-solidifed modified MS (Murashige and Skoog(1962) Physiol. Plant. 15:473-497) medium supplemented with vitamins,2,4-D and glucose. Secondary embryos were placed in flasks in liquidculture maintenance medium and maintained for 7-9 days on a gyratoryshaker at 26+/−2° C. under ˜80 μEm−2s−1 light intensity. The culturemaintenance medium was a modified MS medium supplemented with vitamins,2,4-D, sucrose and asparagine. Prior to bombardment, clumps of tissuewere removed from the flasks and moved to an empty 60×15 mm petri dishfor bombardment. Tissue was dried by blotting on Whatman #2 filterpaper. Approximately 100-200 mg of tissue corresponding to 10-20 clumps(1-5 mm in size each) were used per plate of bombarded tissue.

After bombardment, tissue from each bombarded plate was divided andplaced into two flasks of liquid culture maintenance medium per plate ofbombarded tissue. Seven days post bombardment, the liquid medium in eachflask was replaced with fresh culture maintenance medium supplementedwith 100 ng/ml selective agent (selection medium). For selection oftransformed soybean cells the selective agent used was a sulfonylurea(SU) compound with the chemical name, 2-chloro-N-((4-methoxy-6methy-1,3,5-triazine-2-yl)aminocarbonyl) benzenesulfonamide (commonnames: DPX-W4189 and chlorsulfuron). Chlorsulfuron is the activeingredient in the DuPont sulfonylurea herbicide, GLEAN®. The selectionmedium containing SU was replaced every week for 6-8 weeks. After the6-8 week selection period, islands of green, transformed tissue wereobserved growing from untransformed, necrotic embryogenic clusters.These putative transgenic events were isolated and kept in media with SUat 100 ng/ml for another 2-6 weeks with media changes every 1-2 weeks togenerate new, clonally propagated, transformed embryogenic suspensioncultures. Embryos spent a total of around 8-12 weeks in SU. Suspensioncultures were subcultured and maintained as clusters of immature embryosand also regenerated into whole plants by maturation and germination ofindividual somatic embryos.

In the greenhouse, 1400 T1 plants derived from 42 zag2:ipt::ALStransgenic events were grown at a high density (1400 plants in a spacedesigned for 480). Eighteen plants of variety ‘Jack’ grown in the sameenvironment were used as controls. Plants were grown to maturity andvisually selected for unusual pod clusters, or increased pod load.Eighty three (83) zag2:ipt::ALS plants, and 18 Jack plants were measuredfor the number of pods per plant, number of seed per plant, and seedweight (converted to 100-seed weight). Data were subject to ANOVA usingthe PROC GLM procedure in SAS, and means separation were completed usingthe PROC MEANS function of SAS.

The null hypothesis tested was to determine if zag2:ipt::ALS plantsvisually selected for unusual pod clustering or apparent increased podload were significantly different from the untransformed control (Jack).Plants from 34 events were selected, and the pod number data across allevents selected was significantly different (p=0.05) between the iptplants and Jack. Across all events, the selected zag2:ipt::ALS plantsaveraged 32.1 pods, which was significantly more (LSD=5.2 pods) comparedto the 25.4 pods that Jack averaged.

Five events were identified that were significantly different than Jackat the 0.05 level, and at least 2 plants from each event were measured.When the pod data for these events was subject to ANOVA, thezag2:ipt::ALS plants were statistically different from the Jack plants.The plants from the 5 selected events had an average of 42.3 pods, whichwas statistically greater (LSD=5.9 pods) of the control.

Seed number was counted from all threshed plants of the two events withthe highest average pod number (AFS 3579.7.1 and AFS 3586.1.2). TheZag2:ipt::ALS plants averaged 73.6 seed per plant, which wassignificantly more (LSD=11.7 seed) than average seed per plant of Jack(44.7 seed) (Table 5). The events were not statistically different fromeach other.

Seed of each individual zag2:ipt::ALS plant and individual Jack plantswere weighed to determine if seed size was affected by the increased podload. The 10-seed weight of individual plants from AFS 3579.7.1 and AFS3586.1.2 was 16.6 grams, which was not statistically different (LSD=1.1gram) from the 100 seed weight of the control Jack plants (16.2 grams).

The data examined suggest that the zag2:ipt::ALS construct potentiallymay influence pod number and seed per plant. In addition, seed size forthe zag2:ipt::ALS plants measured was not statistically different fromthe non-transformed Jack control. A high level of variability existed inthe greenhouse environment; however, these preliminary data suggest thatthe zag2:ipt::ALS construct may increase pod retention and seed perplant without a statistical difference in seed size.

Example 13 Isolation of eep1 Promoter Sequences

The procedure for promoter isolation is described in the User Manual forthe Universal Genome Walker kit sold by Clontech Laboratories, Inc.,Palo Alto, Calif. Genomic DNA was prepared by grinding 10-day-old Zeamays seedling leaves in liquid nitrogen, and the DNA prepared using theDNeasy Plant Kit (Qiagen, Valencia, Calif.). The DNA was then usedexactly as described in the Genome Walker User Manual (Clontech PT3042-1version PR68687). Briefly, the DNA was digested separately withrestriction enzymes DraI, EcoRV, PvuII, ScaI, and StuI, all blunt-endcutters. In addition to the blunt enzymes suggested by Clontech, threeother blunt enzymes, EcoICRI, XmnI, and SspI were also used in separatedigestions. The DNA was extracted with phenol, then chloroform, thenethanol precipitated. The Genome Walker adapters were ligated onto theends of the restricted DNA, to create a “Genome Walker Library.”

For isolation of specific promoter regions, two nonoverlappinggene-specific primers (26-30 bp in length) were designed complementaryto the 5′ end of the maize genes identified from sequence databases. Theprimers were designed to amplify the region upstream of the codingsequence, i.e., the 5′ untranslated region and promoter of the chosengene. The sequences of the primers are given below. The first round ofPCR was performed on each Genome Walker library with Clontech primer AP1(SEQ ID NO: 15) and the gene-specific primer (gsp)1 with the sequenceshown in SEQ ID NO: 11.

PCR was performed in a model iCycler thermal cycler from Bio-Rad(Hercules, Calif.) using reagents supplied with the Genome Walker kit.The following cycle parameters were used: 7 cycles of 94° C. for 2seconds, then 68° C. for 3 minutes, followed by 32 cycles of 94° C. for2 seconds and 67° C. for 3 minutes. Finally, the samples were held at67° C. for 4 minutes and then at 4° C. until further analysis.

As described in the User Manual, the DNA from the first round of PCR wasthen diluted and used as a template in a second round of PCR using theClontech AP2 primer (SEQ ID NO: 16) and gene-specific primer (gsp)₂ withthe sequence shown in SEQ ID NO:12.

The cycle parameters for the second round were: 5 cycles of 94° C. for 4seconds, then 70° C. for 3 minutes, followed by 20 cycles of 94° C. for4 seconds, then 68° C. for 3 minutes. Finally, the samples were held at67° C. for 4 minutes and then held at 4° C. Approximately 10 ml of eachreaction were run on 0.8% agarose gel, and bands (usually 500 bp orlarger) were excised, purified with the Qiaquick Gel Extraction Kit(Qiagen, Valencia, Calif.) and cloned into the TA vector pGEMTeasy(Promega, Madison, Wis.). Clones were sequenced for verification.

The band produced from the XmnI Genome Walker library contained 1.5 kbof sequence upstream of the gene specific primer in SEQ ID NO: 12. Theeep1 promoter region was obtained using primers SEQ ID NOS: 13 and 14,created from this sequence to amplify 1 kb of genomic DNA from maizeline A63. These primers added a HindIII site at the 5′ end, an NcoI atthe start of translation, and an EcoRV site just upstream of the NcoIsite. These were added to aid in future vector construction. The PCRreaction was performed in a Bio-Rad iCycler (Hercules, Calif.) thermalcycler using PCR supermix High fidelity (Cat #10790020, Invitrogen,Carlsbad, Calif.). The following cycle parameters were used: 94° C. for2 seconds, followed by 30 cycles of 94° C. for 20 seconds, 55° C. for 30seconds, and 68° C. for 1 minute. Finally, the samples were held at 67°C. for 4 minutes and then at 4° C. until further analysis. The PCRproducts were then cloned into the PGEM-T Easy vector (Promega Corp.Madison, Wis.). Clones were sequenced for verification.

Example 14 Isolation of eep2 Promoter Sequences

The procedure for promoter isolation is described in the User Manual forthe Universal Genome Walker kit sold by Clontech Laboratories, Inc.,Palo Alto, Calif. Genomic DNA was prepared by grinding leaves from Zeamays B73 plants at V6 stage in liquid nitrogen, and the DNA preparedusing the PureGene DNA isolation Kit (Gentra Systems, Minneapolis,Minn.). The DNA was then used exactly as described in the Genome WalkerUser Manual (Clontech PT3042-1 version PR68687). Briefly, the DNA wasdigested separately with restriction enzymes Dra I, which generatesblunt-ends. The DNA was extracted with phenol, then chloroform, followedby ethanol precipitation. The Genome Walker adapters were ligated ontothe ends of the restricted DNA, to create a “Genome Walker Library.”

For isolation of specific promoter regions, two non-overlappinggene-specific primers (27 bp each in length) were designed complementaryto the 5′ end of the maize EST identified from sequence databases. Theprimers were designed to amplify the region upstream of the codingsequence, i.e., the 5′ untranslated region and promoter of the chosengene. The sequences of the primers are given below. The first round ofPCR was performed on the Genome Walker library with Clontech primer AP1(SEQ ID NO: 15) and the gene-specific primer 1 (GSP1) with the sequenceAAACACCTTCGGATATTGCTCCCTTTT (SEQ ID NO: 21).

PCR was performed in a PTC-200 DNA Engine thermal cycler from MJResearch Inc. (Waltham, Mass.) using reagents supplied with the GenomeWalker kit. The following cycle parameters were used: 7 cycles of 94° C.for 10 seconds, then 72° C. for 3 minutes, followed by 32 cycles of 94°C. for 10 seconds and 67° C. for 3 minutes. Finally, the samples wereheld at 67° C. for 7 minutes and then at 8° C. until further analysis.

As described in the User Manual, the DNA from the first round of PCR wasthen diluted and used as a template in a second round of PCR using theClontech AP2 primer (SEQ ID NO: 16) and gene-specific primer 2 (GSP2)with the sequence TCTCGCATTTGCAGAAACGAACAACGT (SEQ ID NO: 22).

The cycle parameters for the second round were: 5 cycles of 94° C. for10 seconds, then 72° C. for 3 minutes, followed by 20 cycles of 94° C.for 10 seconds, then 67° C. for 3 minutes. Finally, the samples wereheld at 67° C. for 7 minutes and then held at 8° C. Approximately 10 μLof each reaction were run on 1.0% agarose gel, and PCR products 500 bpor larger were excised, purified with the Qiaquick Gel Extraction Kit(Qiagen, Valencia, Calif.). The band produced from the Dra I GenomeWalker library contained 1.0 kb of sequence upstream of the GSP2 primer,and it was cloned into the TA cloning vector pCR2.1 (Invitrogen,Carlsbad, Calif.). Clones were sequenced for verification. The eep2promoter region was obtained by PCR from the plasmid using primerscorresponding to a 1027 bp region from downstream of AP2 primer andupstream of the ATG start codon. Clones were sequenced for verification.

The EST distribution for eep2 is as follows:

p0083.cldeu53r B73 “Kernel” “” “7 DAP whole kernels” p0124.cdbmq47r B73“Kernel, Embryo” “” “6 day embryo sac, Screened 1” p0062.cymab46r B73“Kernels, Endosperm” “” “coenocytic (4 DAP) embryo sacs,” p0106.cjlps68rB73 “Kernel” “” “5 DAP whole kernels, screened 1” p0124.cdbmq21r B73“Kernel, Embryo” “” “6 day embryo sac, Screened 1” p0100.cbaab57r B73“Kernel, Embryo, Endosperm” “” “coenocytic (4 DAP) embryo sacs, screened1 (original lib P0062)” p0100.cbaac19r B73 “Kernel, Embryo, Endosperm”“” “coenocytic (4 DAP) embryo sacs, screened 1 (original lib P0062)”p0062.cymal89r B73 “Kernels, Endosperm” “” “coenocytic (4 DAP) embryosacs,” p0062.cymai74f B73 “Kernels, Endosperm” “” “coenocytic (4 DAP)embryo sacs,”Lynx data for eep2 in PPM:

PPM Name Adj Title Cen6lm 10261 B73 endosperm, 6 DAP embryo sac Cdk8lm457 Corn whole kernels, embryo and endosperm, 8 DAP Cpd1-ctr 395 Cornpedicels control Cpd1-drg 375 Corn pedicels drought-stressed Cen8lm 312Corn endosperm 8 DAP Oper5lm 8 B73, 5 DAP pericarp Cebho4lm 5 Cornembryos Askc0, 15 DAP Cen12lm 2 Corn endosperm 12 DAPThese data are very consistent with limiting this gene's expression tothe developing seed.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationscan be practiced within the scope of the appended claims.

1. An isolated promoter which drives transcription in a seed-preferredmanner, wherein the promoter comprises the nucleotide sequence set forthin SEQ ID NO:
 7. 2. A recombinant expression cassette comprising apromoter and a nucleotide sequence operably linked to said promoter,wherein said promoter comprises the nucleotide sequence set forth in SEQID NO:
 7. 3. A plant stably transformed with the expression cassette ofclaim 2.